Abstract: Proteomic analysis was conducted on pepper S 033-A maintainer line and S 033-B near-isogenic line to reveal the key proteins of pepper sterility. The results showed that the total number of identified proteins in maintainer line and sterile line was 7667, the quantified proteins were 7629, and there were 504 differentially expressed proteins. Out of these, 351 proteins was upregulated expression in the maintainer line S 033-A, and 153 ones was upregulated expression in the sterile line S 033-B. Combined with bioinformatics, subcellular localization, conserved domain, GO and KEGG enrichment analysis of differentially expressed proteins were performed. It was found that subcellular localization was mainly in cytoplasm, nucleus and chloroplast, of which 421 proteins had conserved domains, which mainly were glutathione S-transferase, peroxidase, UDP-glucoside and UDP-glucoside transferase. GO enrichment was found within chloroplast, plastid and thylakoid. KEGG enrichment analysis showed that it was mainly involved in photosynthesis, cytochrome P 450, monoterpene biosynthesis and flavonoid biosynthesis pathways. In this study, we found the related key protein and metabolic pathways of pepper male sterility from the perspective of proteomics, and revealed the possible molecular mechanism of pepper male sterility lines and identified related candidate protein, which laid a theoretical foundation for pepper genetics and breeding.