Objective  To explore the effect of different modes of blood flow shear stress on the Pim1 expression in human umbilical vein endothelial cells (HUVECs) and the regulation of phosphorylation at Ser1177. and Ser633 of endothelial nitric oxide synthase (eNOS).     Methods  HUVECs were isolated from fresh human umbilical cord. The parallel plate flow chamber system was used to load 15 dyn/cm2 laminar shear stress (LSS) and (0.5 ± 4) dyn/cm2 oscillatory shear stress (OSS) on HUVECs. Western blotting was used to evaluate the protein levels of Pim1, phosphorylated Akt at Ser473, and phosphorylated eNOS at Ser633 and Ser1177. Small interfering RNA (siRNA) was used to knockdown of Pim1 and Akt.     Results  LSS obviously induced Pim1 protein expression in HUVECs (P<0.01) compared with OSS stimulation. LSS also significantly increased Akt phosphorylation at Ser473 and eNOS phosphorylation both at Ser633 and Ser1177 (P<0.05 or P<0.01). Pim1 silencing, or treatment with SMI-4a, an inhibitor of Pim1, abolished the above effects of LSS-stimulated HUVECs (P<0.05 or P<0.01). Knockdown of Akt also prevented the LSS-induced Akt phosphorylation at Ser473 and eNOS phosphorylation both at Ser1177 and Ser633 (P<0.05 or P<0.01). However, Akt knockdown did not alter the expression of Pim1 induced by LSS.     Conclusion  Shear stress can regulate the phosphorylation of eNOS Ser633 and Ser1177 in HUVECs through the Pim1/Akt signaling pathway.