Background; During mitosis and meiosis, centromere proteins (CENPs) play a key role in proper chromosome segregation. Abnormal expression of CENPs leads to chromosome instability, which is the main cause of tumorigenesis. Methods; To elucidate the functional characteristics of CENPs in pan-cancer, we comprehensively analyzed the expression landscape of CENPs and their relationships with patient survival, genomic alterations, tumor immunity, tumor microenvironment, and anticancer drug sensitivity. The expression patterns and signaling pathways of CENPs were identified through multiple bioinformatics mining and experimental verification. GEPIA2 and PrognoScan were implemented to evaluate the prognostic value of CENPs. The molecular functions of CENPs in pan-cancer were comprehensively assessed using cBioPortal, GSCA, ImmuCellAI, CellMiner, the ROC plotter tool and TIDE. Results; The results showed that CENPs were upregulated in most tumors compared with normal tissues. We confirmed this conclusion by immunohistochemistry and real-time quantitative PCR. Survival analysis revealed a significant association between high CENP expression and a poor prognosis. CENP expression is related to genome alterations, copy number variation, single nucleotide variation and methylation. Among CENP family genes, CENPF and CENPE are mutated at high frequencies in various tumors, while CENPM and CENPA are less frequently mutated. Furthermore, CENPs regulate the tumor mutational burden, stemness, and microsatellite instability, and are associated with tumor immunity. Most importantly, we revealed that CENP family gene expression was correlated with chemosensitivity and immunotherapy responses. Conclusion; These findings may clarify the role of CENPs in cancer progression and antitumor drug sensitivity and provide evidence for CENPs as a potential target in pan-cancer.