BACKGROUND: nuclear-associated antigen Ki67 (Ki67) emerges as a clinically practical biomarker for proliferation assessment among many cancer types. However, the definite prognostic value of Ki67 against a specific cancer type has remained vague. This study aims to perform a comprehensive pan-cancer analysis of the prognosis value of Ki67 across various cancer types. METHODS: This study explored the expression, prognostic value, and tumor-infiltrating immune of MKI67 in the TCGA database by pan-cancer, and then performed immunohistochemical, correlation analysis and prognostic analysis using 10028 patients of the top 10 cancer patients in China we collected. The correlation between MKI67 expression and survival outcome, clinical features, MSI, TMB, and tumor-infiltrating immune cells by TCGA database, xCell, and TIMER algorithms. RESULTS: MKI67 expression was significantly upregulated across varied cancer types verified by datasets. We found MKI67 expression was significantly associated with poor prognosis in LUADLUSC, LIHC, and BRCA patients, but good prognosis in COADREAD and READ patients via Kaplan-Meier survival analysis using 10028 patients collected. These results of our validation were generally consistent with TCGA database except BRCA, COADREAD and READ. Meanwhile, upregulation of MKI67 elevates the degree of immune infiltration of several immune cell subtypes, such as functional T cells, CD4 CONCLUSION: Our comprehensive analysis may supply useful guidance on MKI67 applicability across various cancer types. These observed results contribute to the promise of MKI67 in a realistic clinical setting and improve the outcomes of cancer patients.