<p><strong>Abstract.</strong> Coexisting plant species in a karst ecosystem may use diverse strategies of trade off between carbon gain and water loss to adopt to the low soil nutrient and low water availability conditions. An understanding of the impact of <span class="inline-formula">CO<sub>2</sub></span> diffusion and maximum carboxylase activity of Rubisco (<span class="inline-formula"><i>V</i><sub>cmax</sub></span>) on the light-saturated net photosynthesis (<span class="inline-formula"><i>A</i></span>) and intrinsic water use efficiency (iWUE) can provide insight into physiological strategies of the water–carbon regulation of coexisting plant species used in adaptation to karst environments at the leaf scale. We selected 63 dominant species (across 6 life forms) in a subtropical karst primary forest in southwestern China, measured their <span class="inline-formula">CO<sub>2</sub></span> response curves, and calculated the corresponding stomatal conductance to <span class="inline-formula">CO<sub>2</sub></span> <span class="inline-formula">(<i>g</i><sub>s</sub>)</span>, mesophyll conductance to <span class="inline-formula">CO<sub>2</sub></span> <span class="inline-formula">(<i>g</i><sub>m</sub>)</span>, and <span class="inline-formula"><i>V</i><sub>cmax</sub></span>. The results showed that <span class="inline-formula"><i>g</i><sub>s</sub></span> and <span class="inline-formula"><i>g</i><sub>m</sub></span> varied about 7.6- and 34.5-fold, respectively, and that <span class="inline-formula"><i>g</i><sub>s</sub></span> was positively related to <span class="inline-formula"><i>g</i><sub>m</sub></span>. The contribution of <span class="inline-formula"><i>g</i><sub>m</sub></span> to the leaf <span class="inline-formula">CO<sub>2</sub></span> gradient was similar to that of <span class="inline-formula"><i>g</i><sub>s</sub></span>. <span class="inline-formula"><i>g</i><sub>s</sub></span><span class="thinspace"></span><span class="inline-formula">∕</span><span class="thinspace"></span><span class="inline-formula"><i>A</i></span>, <span class="inline-formula"><i>g</i><sub>m</sub></span><span class="thinspace"></span><span class="inline-formula">∕</span><span class="thinspace"></span><span class="inline-formula"><i>A</i></span> and <span class="inline-formula"><i>g</i><sub>t</sub></span><span class="thinspace"></span><span class="inline-formula">∕</span><span class="thinspace"></span><span class="inline-formula"><i>A</i></span> was negatively related to <span class="inline-formula"><i>V</i><sub>cmax</sub></span><span class="thinspace"></span><span class="inline-formula">∕</span><span class="thinspace"></span><span class="inline-formula"><i>A</i></span>. The relative limitations of <span class="inline-formula"><i>g</i><sub>s</sub></span> <span class="inline-formula">(<i>l</i><sub>s</sub>)</span>, <span class="inline-formula"><i>g</i><sub>m</sub></span> (<span class="inline-formula"><i>l</i><sub>m</sub>)</span>, and <span class="inline-formula"><i>V</i><sub>cmax</sub></span> (<span class="inline-formula"><i>l</i><sub>b</sub>)</span> to <span class="inline-formula"><i>A</i></span> for the whole group (combined six life forms) were significantly different from each other (<span class="inline-formula"><i>P</i></span><span class="thinspace"></span>&lt;<span class="thinspace"></span>0.05). <span class="inline-formula"><i>l</i><sub>m</sub></span> was the largest (0.38<span class="thinspace"></span><span class="inline-formula">±</span><span class="thinspace"></span>0.12), followed by <span class="inline-formula"><i>l</i><sub>b</sub></span> (0.34<span class="thinspace"></span><span class="inline-formula">±</span><span class="thinspace"></span>0.14), and <span class="inline-formula"><i>l</i><sub>s</sub></span> (0.28<span class="thinspace"></span><span class="inline-formula">±</span><span class="thinspace"></span>0.07). No significant difference was found between <span class="inline-formula"><i>l</i><sub>s</sub></span>, <span class="inline-formula"><i>l</i><sub>m</sub></span>, and <span class="inline-formula"><i>l</i><sub>b</sub></span> for trees and tree/shrubs, while <span class="inline-formula"><i>l</i><sub>m</sub></span> was the largest, followed by <span class="inline-formula"><i>l</i><sub>b</sub></span> and <span class="inline-formula"><i>l</i><sub>s</sub></span> for shrubs, grasses, vines and ferns (<span class="inline-formula"><i>P</i></span><span class="thinspace"></span>&lt;<span class="thinspace"></span>0.05). iWUE varied about 3-fold (from 29.52 to 88.92<span class="thinspace"></span><span class="inline-formula">µ</span>mol<span class="thinspace"></span><span class="inline-formula">CO<sub>2</sub></span><span class="thinspace"></span>mol<span class="inline-formula"><sup>−1</sup></span><span class="thinspace"></span><span class="inline-formula">H<sub>2</sub>O</span>) across all species, and was significantly correlated with <span class="inline-formula"><i>g</i><sub>s</sub></span>, <span class="inline-formula"><i>V</i><sub>cmax</sub></span>, <span class="inline-formula"><i>g</i><sub>m</sub></span><span class="thinspace"></span><span class="inline-formula">∕</span><span class="thinspace"></span><span class="inline-formula"><i>g</i><sub>s</sub></span>, and <span class="inline-formula"><i>V</i><sub>cmax</sub></span><span class="thinspace"></span><span class="inline-formula">∕</span><span class="thinspace"></span><span class="inline-formula"><i>g</i><sub>s</sub></span>. These results indicated that karst plants maintained relatively high <span class="inline-formula"><i>A</i></span> and low iWUE through the covariation of <span class="inline-formula"><i>g</i><sub>s</sub></span>, <span class="inline-formula"><i>g</i><sub>m</sub></span>, and <span class="inline-formula"><i>V</i><sub>cmax</sub></span> as an adaptation to a karst environment.</p>