Aim To explore the pyroptosis of vascular smooth muscle cell (VSMC) induced by Chlamydia pneumoniae (C.pn) infection and its possible mechanisms. Methods Primary rat VSMC were cultured by explant method. After the model of VSMC infected with C.pn was established, the changes in morphology of VSMC were observed under an inverted phase microscope, the lactic dehydrogenase (LDH) content was detected by the kit, and the expression levels of GSDMD and Caspase-1 were determined by Western blot, the changes in mitochondrial oxidative phosphorylation and the expression of complex-related proteins were measured by quantitative proteomic analysis by tandem mass tag technology and gene ontology. Results Compared with the control group, bubble-like vesicles were found outside the membrane of VSMC after C.pn infection under an inverted phase microscope. After C.pn infection of VSMC for 36 h and 48 h, LDH content increased by 38.92% and 79.54% (P＜0.001), respectively, and the expression of pyropotosis-related protein GSDMD increased by 1.74 times and 1.67 times (P＜0.001). After C.pn infection of VSMC for 48 h, the expression(pro-Caspase-1) and activity(Caspase-1 p12/p10)of Caspase-1 increased by 2.69 times and 3.47 times (P＜0.001), respectively. The mass spectrometry results showed that there were 20 differentially expressed proteins enriched in the oxidative phosphorylation pathway after C.pn infection, and at the same time, ComplexⅠubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4) decreased significantly. Further Western blot results showed that the expression level of NDUFS4 decreased by 57.5% and 57% (P＜0.001) after C.pn infection of VSMC for 36 h and 48 h respectively. Conclusion C.pn infection may induce VSMC pyroptosis by affecting mitochondrial function through downregulating NDUFS4 expression.