Objective To investigate the roles of protein phosphatase 2C (PP2C) activated by palmitic acid (PA) in the phosphorylation modulation of endothelial nitric oxide (eNOS) at the site of serine 1177 (eNOS Ser1177) in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were randomly divided into control group, PA group, specific PP2C inhibitor sanguinarine (San) combined with PA group and PP2Cα specific small interference RNA (siRNA) transfection group. The protein expression of total eNOS, phosphorylated eNOS Ser1177 and PP2Cα were detected by Western blotting. The intracellular NO content was measured by DAF-FM DA. The co-localization of eNOS protein and PP2C protein was observed by co-immunoprecipitation. Results Compared with the control group, the phosphorylation levels of eNOS Ser1177 and NO content decreased significantly in the PA group. The PP2C inhibitor sanguinarine reversed PA-induced decrease of eNOS Ser1177 phosphorylation level and NO content. The phosphorylation levels of eNOS Ser1177 were enhanced dramatically after PP2Cα protein was knocked down by specific siRNA. The eNOS protein and PP2C protein co-localized in endothelial cells. Conclusion PA reduces the phosphorylation level of endothelial eNOS Ser1177 in HUVECs by activating PP2C.