Cyclin-dependent kinase 5 (CDK5) plays a critical role in the inflammatory response. Macrophages are pivotal orchestrators of inflammation, fibrosis, and wound repair. However, the effectiveness of CDK5 in macrophages on cutaneous wound healing remains inadequately characterized. We determined the role of CDK5 signaling pathway in macrophages in mouse cutaneous wound healing through the established macrophage-specific deletion of CDK5 (myeCDK5-/-) mice and the pharmacological CDK5 inhibitor Roscovitine. Phosphorylated proteomics, western blotting, Masson staining, and dualimmunofluorescence staining were performed to investigate the potential mechanisms underlying CDK5-mediated inflammatory regulation in macrophages in wound healing. CDK5 expression and phosphorylation were both elevated significantly in cutaneous wound healing process in mice. Moreover, an accelerated wound healing in myeCDK5-/- mice was exhibited with the reduced pro-inflammatory mediators (IL-1β and iNOS) and the elevated anti-inflammatory markers (IL-10 and CD163) expression significantly. CDK5 deficiency in macrophages enhanced tissue remodeling, evidenced by increased collagen deposition and capillary density (CD31+ cells). Consistently, Roscovitine-treated mice also showed accelerated wound healing, accompanied by decreased pro-inflammatory factors and increased anti-inflammatory markers at the wound site. Mechanistically, the decreased phosphorylation of SIRT1 at the Ser14 and Ser47 sites, as a substrate of CDK5, was confirmed in myeCDK5-/- mice. These data are the first to indicate that CDK5 signaling-dependent regulation of SIRT1 phosphorylation in macrophage-mediated inflammation is required for the wound healing process, warranting consideration of the CDK5-SIRT1 pathway as a therapeutic target for cutaneous wound healing.© 2025. The Author(s).