The aim of the present study was to examine the effect of small interfering RNA (siRNA) methods on the expression of N­ethylmaleimide sensitive factor (NSF) and Weibel­Palade body (WPB) release in endothelial cells. A small hairpin RNA (shRNA), mediated with an adenovirus vector, was designed to target the N­terminal functional area of NSF. Subsequently, viruses were transfected into human aortic endothelial cells. The mRNA and protein expression levels of NSF were detected using reverse transcription­quantitative polymerase chain reaction and Western blot analyses, respectively, and the release of WPBs in the endothelial cells was examined using immunofluorescence. The mRNA expression of NSF in the endothelial cells, which were transfected with the adenoviruses carrying the NSF­shRNA was significantly decreased, compared with the negative control group (P=0.035) and blank control group (P=0.02). In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05). The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249). The immunofluorescence staining showed that the release of WPBs in the endothelial cells induced with thrombin was inhibited markedly following transfection with the virus carrying the NSF­shRNA. Therefore NSF­siRNA inhibited the mRNA and protein expression levels of NSF, and inhibited the release of WPBs in endothelial cells induced with thrombin. These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome.