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Persistent inflammation impedes wound healing due to the excessive infiltration of inflammatory cells and the accumulation of pro-inflammatory cytokines. Emu oil (EO) exhibits strong anti-inflammatory properties, but the molecular mechanisms underlying the anti-inflammatory effects on wound healing remain largely unknown. Emu oil promotes wound healing by accelerating the wound closure rate, re-epithelialization, and collagen formation. In vivo, emu oil enhanced the secretion of IL-10 while suppressing IL-1β secretion, promoting the M2 macrophage polarization, and significantly reducing the phosphorylation of JNK and p38. In vitro, emu oil decreased the expression of IL-1β and TNF-α in the RAW264.7 inflammation model while inhibiting the activation of p-JNK and p-p38. The immunomodulatory effect of emu oil was found to be mediated via the suppression of JNK signaling, leading to enhanced anti-inflammatory responses and pro-polarization of M2 macrophages. These findings provide valuable and innovative insights into the therapeutic potential of emu oil in wound healing.