The development of biotechnological lactic acid production has attracted attention to the potential production of an optically pure isomer of lactic acid, although the relationship between fermentation and the biosynthesis of highly optically pure d-lactic acid remains poorly understood. Sporolactobacillus terrae SBT-1 is an excellent d-lactic acid producer that depends on cultivation conditions. Herein, three enzymes responsible for synthesizing optically pure d-lactic acid, including d-lactate dehydrogenase (D-LDH; encoded by ldhDs), l-lactate dehydrogenase (L-LDH; encoded by ldhLs), and lactate racemase (Lar; encoded by larA), were quantified under different organic nitrogen sources and concentration to study the relationship between fermentation conditions and synthesis pathway of optically pure lactic acid. Different organic nitrogen sources and concentrations significantly affected the quantity and quality of d-lactic acid produced by strain SBT-1 as well as the synthetic optically pure lactic acid pathway. Yeast extract is a preferred organic nitrogen source for achieving high catalytic efficiency of d-lactate dehydrogenase and increasing the transcription level of ldhA2, indicating that this enzyme plays a major role in d-lactic acid formation in S. terrae SBT-1. Furthermore, lactate racemization activity could be regulated by the presence of d-lactic acid. The results of this study suggest that specific nutrient requirements are necessary to achieve a stable and highly productive fermentation process for the d-lactic acid of an individual strain.