OBJECTIVE： To study the effects of alprostadil on the expression of vascular endothelial growth factor （VEGF） and endothelial nitric oxide synthase （eNOS） in human pulmonary microvascular endothelial cells （HPMECs） with hypoxia-induced injury. METHODS： HPMECs were divided into group A （normal cultural environment）， group B （3％O2）， group C （3％O2+15 μg/L alprostadil） and group D （3％O2+45 μg/L alprostadil） for 24 h culture. Cell morphology was observed； MTT method was conducted to detect cell activity （recorded by absorbance）； flow cytometry was adopted to detect apoptosis rate； Western blot was used to detect relative expressions of VEGF and eNOS in cells. RESULTS： Cell density in group A and D was relatively high， that in group B and C was low. Compared with group A， cell activities decreased and apoptosis rates increased in group B， C and D， expressions of VEGF and eNOS enhanced， eNOS/VEGF decreased （P＜0.05）. Compared with group B， cell activities increased in group C and D， VEGF expression decreased （P＜0.05）； apoptosis rate in group D decreased， eNOS expression decreased and eNOS/VEGF increased. Compared with group C， cell apoptosis rate was decreased in group D， VEGF expression decreased and eNOS/VEGF increased （P＜0.05）. CONCLUSIONS： Alprostadil maybe play effect on HPMECs with hypoxia-induced injury by up-regulating the expression level of eNOS/VEGF.