Inflammation and microenvironment play a crucial role in muscle regeneration. IL (interleukin)-6, as a multifunctional cytokine is involved in the processes. However, the causative effect of IL-6 in muscle regeneration remains unclear. In a mouse model of cardiotoxin-induced muscle injury/regeneration, infiltrated monocytes/macrophages produce a high level of IL-6 started on 1 day (24 h) after injury. In IL-6 knock-out (−/−) mice, the muscle regeneration procedure was impaired along with decreased myogenic determination factor (MyoD) and myogenin mRNA level and increased interstitial fibrosis. The IL-6−/− mice exhibited less macrophage infiltration, lower inflammatory cytokine (IL-1β, inducible NO synthase, Transforming growth factor (TGF)-β1, and IL-10) and chemokine (CCL2, CCL3, and CCL5) expression, and inhibited myoblast proliferation. In vitro, IL-6 deficiency or Signal Transducer and Activator of Transcription 3 (STAT3) knockdown in activated macrophage attenuated the expression of CCL2, CCL3, but not CCL5, which resulted in less macrophage migration. Moreover, inflammatory macrophages promoted myoblast proliferation in an IL-6-dependent manner. Finally, adoptive transfer IL-6+/+ BM cells into IL-6−/− mice rescued the impaired regeneration with improved MyoD and myogenin expression. Taken together, IL-6 expression and the activated STAT3 signaling pathway in monocytes/macrophages is a critical mediator of macrophage migration and myoblast proliferation during muscle regeneration. Background: Interleukin-6 (IL-6), as a multifunctional cytokine, was involved in the inflammation microenvironment of muscle regeneration. Results: In mice lacking IL-6, less macrophage recruitment and decreased myoblast proliferation impairs muscle regeneration. Conclusion: In monocytes/macrophages, activation of the IL-6/STAT3 pathway is critical to macrophage migration and myoblast proliferation during muscle regeneration. Significance: IL-6/STAT3 pathway is essential for muscle regeneration.