This study investigated the changes in human umbilical vein endothelial cells (HUVECs) induced by overexpression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor VIII-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the proliferative ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS (eNOS) activity was assayed by using spectrophotometry, and NO2 (-)/NO3 (-) levels were measured using nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma membrane and cytoplasm, and Western blot analysis confirmed that NOSTRIN levels were significantly higher in cells transfected with the NOSTRIN plasmid (P < 0.01). The activity of eNOS and the levels of NO2 (-)/NO3 (-) were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells (P < 0.01 and P < 0.01, respectively). Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOSTRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overexpression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.