Vascular hyporeactivity is an important factor in irreversible shock, and post-shock mesenteric lymph (PSML) blockade improves vascular reactivity after hemorrhagic shock. This study explored the possible involvement of myosin light chain kinase (MLCK) in PSML-mediated vascular hyporeactivity and calcium desensitization. Rats were divided into sham (n=12), shock (n=18), and shock+drainage (n=18) groups. A hemorrhagic shock model (40±2 mmHg, 3 h) was established in the shock and shock+drainage groups. PSML drainage was performed from 1 to 3 h from start of hypotension in shock+drainage rats. Levels of phospho-MLCK (p-MLCK) were determined in superior mesenteric artery (SMA) tissue, and the vascular reactivity to norepinephrine (NE) and sensitivity to Ca 2+ were observed in SMA rings in an isolated organ perfusion system. p-MLCK was significantly decreased in the shock group compared with the sham group, but increased in the shock+drainage group compared with the shock group. Substance P (1 nM), an agonist of MLCK, significantly elevated the decreased contractile response of SMA rings to both NE and Ca 2+ at various concentrations. Maximum contractility (E max) in the shock group increased with NE (from 0.179±0.038 to 0.440±0.177 g/mg, P<0.05) and Ca 2+ (from 0.515±0.043 to 0.646±0.096 g/mg, P<0.05). ML-7 (0.1 nM), an inhibitor of MLCK, reduced the increased vascular response to NE and Ca 2+ at various concentrations in the shock+drainage group (from 0.744±0.187 to 0.570±0.143 g/mg in E max for NE and from 0.729±0.037 to 0.645±0.056 g/mg in E max for Ca 2+, P<0.05). We conclude that MLCK is an important contributor to PSML drainage, enhancing vascular reactivity and calcium sensitivity in rats with hemorrhagic shock.