Mixed lineage kinase domain-like protein (MLKL), a critical necroptosis effector, is strongly linked to inflammation, a key component of skin wound healing. However, its precise role in the wound healing process remains inadequately characterized. This study revealed sustained MLKL overexpression throughout the wound healing process, not limited to the early inflammation phase. Wound healing was delayed in MLKL-deficient (MLKL) mice compared to wild type C57BL/6J (MLKL) mice, with impaired morphological and pathological recovery. MLKL deficiency reduced the synthesis of inflammatory factors (IL-6, TNF-α, PGE), tissue repair molecules (EGF, VEGF, ERα, MMP-9), and apoptosis markers (Caspase-3, p53, Bcl-2) at wound site. Subsequently, a co-culture system was established to explore the roles of MLKL in macrophage-fibroblast interactions. M1 or M2 macrophages (M1ø or M2ø) were co-cultured with fibroblast-conditioned medium (MFbCM), and fibroblasts were co-cultured with macrophage-conditioned medium (M1ø CM or M2ø CM). The results indicated that MLKL M1ø CM and M2ø CM significantly increased ERα, VEGF and MMP-9 protein expression levels in fibroblasts, whereas this effect was impaired when MLKL M1ø CM or M2ø CM were used. Similarly, MLKL MFbCM upregulated IL-6, NO, and TNF-α in M1ø and IL-10, arginase, and Ym-1 in M2ø, but these effects were diminished with MLKL MFbCM treatment. These results indicate bidirectional crosstalk between macrophages and fibroblasts, in which MLKL plays a role. Additionally, PGE was identified as a downstream mediator of MLKL-mediated interactions between macrophages and fibroblasts. In conclusion, these findings identify MLKL as a multifunctional regulator in skin wound healing in mice.