Sirtuin1 (SIRT1) is a mammalian NAD+-dependent type III deacetylase that plays paramount roles in diverse cellular processes. The nucleocytoplasmic shuttling of SIRT1 was discovered more than a decade ago, but the roles of subcellular SIRT1 localization in tumor progression remain unclear. Here, we report that cytoplasmic SIRT1 acts as a tumor suppressor in ovarian carcinoma. By creating ovarian carcinoma cell lines overexpressing wild-type SIRT1 and nuclear localization signals (NLSs) mutated SIRT1 together with both unbiased proteomic and acetylomic approaches and Transwell assays, we identified that mutations in the NLS sequences prevented SIRT1 from entering the nucleus, resulting in the predominant cytoplasmic localization of SIRT1; the cytoplasmic localization of SIRT1 suppressed the mesenchymal program, activated the epithelial program, and inhibited the migration and invasion of tumor cells, thus providing experimental evidence that SIRT1 functions as a tumor suppressor or oncogene may depend on its subcellular localization. Altogether, our findings may highlight a novel role of cytoplasmic SIRT1 in ovarian carcinoma, providing new possible insights for studies investigating the role of SIRT1 in tumor progression.