BACKGROUND: S-phase kinase-associated protein 2 (SKP2) is a typical oncogene aberrantly overexpressing in a variety of cancer types, but it remains elusive whether SKP2 regulates the antitumor immunity of triple-negative breast cancer. METHODS: The efficacy of anti-PD-1 was evaluated in the orthotopic xenografts of immunocompetent mice models. The infiltration of cytotoxic T cells in tumor microenvironment(TME) were assessed by immunofluorescence staining. The levels of pro-inflammatory chemokines were analyzed by ELISA. The protein interaction was analyzed by co-immunoprecipitation and GST pull-down. The genomic instability was analyzed by fluorescent microscopy. RESULTS: SKP2 inhibition significantly improved the antitumor efficacy of immune checkpoint blockade (ICB). Furthermore, SKP2 inhibition activated the cGAS/STING signal pathway and induced the secretion of pro-inflammatory chemokines, thereby promoting cytotoxic T cell infiltration. Additionally, we identified CDC6, a DNA replication licensing factor as a novel substrate of SKP2 in addition to CDT1. SKP2 induced protein degradation of CDC6 and CDT1 through the ubiquitin-proteasome pathway. Conversely, SKP2 inhibition elevated CDC6 and CDT1 protein levels, which caused DNA aberrant replication, DNA damage and genomic instability, thereby resulting in the accumulation of cytosolic DNA, activating cGAS/STING signaling pathway and improving antitumor immunity. CONCLUSION: SKP2 may be used as an effective therapeutic target to enable ICB antitumor immunotherapy. SOCIAL MEDIA: Peng et al. found that SKP2 inhibition improved the antitumor immunotherapy by activating tumor cell-intrinsic immunity, thereby providing evidences that SKP2 may be used as an effective therapeutic target to enable ICB antitumor immunotherapy.