The immune system is not only essential for host defense but also involved in tissue maintenance and disease pathogenesis. Macrophages play a key role in tissue repair, fibrosis and tumorigenesis, but the mechanisms underlying their multi-functionality have been poorly explored. Here, we identified Mrep (Ly6ChighCX3CR1lowPDPN+CD9+), as a crucial subset of macrophages for muscle regeneration after muscle injury. Muscle regeneration required Mrep-derived activin A, which was produced via TLR4-TIR-domain-containing adapter-inducing interferon-beta (TRIF)-TANK-binding kinase 1 (TBK1)-Interferon regulatory factor (IRF)3/7 signaling pathway in response to muscle injury. Mrep exerted pathological effects by secreting activin A in a model of genetically induced heterotopic ossification (HO), which was suppressed by the TLR4 inhibition. Thus, this study explains the context-dependent functions of macrophages and the link between injury and HO, suggesting that Mrep serves as a potential therapeutic target for regenerating muscles and suppressing HO.