Background: It has been reported that loss of sirtuin 6 (Sirt6) exhibit impaired healing in skin wounds. Given the heterogeneity of macrophage phenotypes during wound repair and the regulation of macrophage phenotypes by sirtuins, we here hypothesized that Sirt6 deletion in myeloid cells would affect macrophages phenotypes in skin wounds and delay wound closure. Objectives: To find out the role of Sirt6 regarding macrophage phenotype in wound healing. Methods: To address this question, floxed Sirt6 mice were bred with lysozyme M-Cre mice to generate myeloid Sirt6 knockout (KO) mice with reduced Sirt6 expression. A full-thickness excisional lesion was made on the dorsal skin of the mice. Results: Compared with wild type mice, wound closure was delayed in KO mice with less collagen deposition, attenuated vWF expression and reduced expression of wound healing related genes. Using immunohistochemistry and RNA expression analyses on macrophage subpopulations from wound tissues, we identified an increased infiltration of M1 type macrophages in KO mice compared with wild type mice. Consistent with the in vivo defects in wound closure, the in vitro study demonstrated that keratinocytes and fibroblasts treated with KO macrophage conditioned medium migrated slower than those of wild type. Conclusion: These results suggest that an imbalance of macrophage phenotypes by Sirt6 deletion contributes to impaired wound healing in mice.