Objective To investigate the expression of charged multivesicular body proteins 5(CHMP5) in pyroptosis of human umbilical vein endothelial cell (HUVEC) and the effect of knocking down CHMP5 on HUVEC pyroptosis. Methods Using polyinosinic-polycytidylic acid (Poly I:C) to stimulate HUVEC to establish an in vitro model of double-stranded RNA (dsRNA) induced HUVEC injury in viral sepsis. HUVEC were randomly divided into control, Lipo, Poly I:C, siNC and siCHMP5+Poly I:C groups, and the expression of CHMP5 in each group was detected by real-time fluorescent quantitative PCR (qRT-PCR) to verify the transfection efficiency. Lactate dehydrogenase (LDH) cytotoxicity assay kit was used to detect the release rate of LDH, ELISA was used to detect the secretion of interleukin-1β(IL-1β) and transmission electron microscopy (TEM) was used to detect the ultrastructure of HUVEC to observe the integrity of the cell membrane, and Western blotting was used to detect the expression level of the cleaved caspase-3(Casp-3) and gasdermin E N-terminal(GSDME-N), and immunofluorescence staining was used to detect the expression and localization of the related proteins in each group of cells. Results In comparison with the control group, the HUVEC in the Poly I:C group were swollen and showed multiple cell membrane ruptures, and cleaved Casp-3, GSDME-N and CHMP5 protein expression was up-regulated. Compared with the control group and the siNC group, knocking down CHMP5 by RNAi technology resulted in increased release of LDH and IL-1β, up-regulation of the expression level of pyroptosis-related proteins, alteration of the distribution of GSDME protein recruitment, and significant aggravation of endothelial cell pyroptosis in the siCHMP5+Poly I:C group. Conclusions The CHMP5 is highly expressed in HUVEC pyroptosis. Knockdown of CHMP5 can enhances HUVEC cellular pyroptosis, which may be related to the inhibition of membrane repair and promoting secondary cellular pyroptosis induced by cleavage of GSDME and GSDMD proteins.