Abstract: OBJECTIVE To observe the inhibition effect of neoliensinine (Neo) on the contraction of mesenteric vascular smooth muscle and vascular smooth muscle cells (VSMCs) of mice， and to explore the mechanism of the effect by observing phosphorylation of myosin light chain (MLC₂₀) in vascular smooth muscle and expression of ROCK1 (RhoA associated protein kinase) in VSMCs. METHODS The microvascular tension tester was used to evaluate the inhibition effect of Neo pretreatment on mesenteric vessel contraction stimulated by high potassium solution， and Urea/glycerol -PAGE was applied to detect its change of phosphorylation of MLC₂₀. Meanwhile， image analyzer was used to detect the effect of Neo on VSMCs contraction induced by high potassium solution， and cell immunofluorescence assay was adopted to detect its expression of ROCK1 protein in VSMCs. RESULTS Neo had a significant inhibitory effect on the contraction of mesenteric vessel induced by high potassium solution with IC₅₀ of 6.019 μmol/L and 95% confidence intervals from 5.299～6.838 μmol/L， and could block the phosphorylation of MLC₂₀ induced by high potassium. In addition， compared with the VSMCs length of model group (KCl) (40.94±1.94)μm， that of treatment group (Neo+KCl) were prolonged (44.95±5.15)μm significantly (P<0.01)， and Neo reduced the expression of ROCK1 protein in VSMCs. CONCLUSION Neo could inhibit the contraction of mesenteric vascular smooth muscle， and its mechanism might be related to blocking the phosphorylation of smooth muscle MLC₂₀ and down-regulating the expression of ROCK1 protein in VSMCs.