Mechanical stress has been recognized as a key inducer of bone regeneration in bone damage, which is experimentally mimicked by distraction osteogenesis (DO), a bone-regenerative process induced by post-osteotomy distraction of the surrounding vascularized bone segments, and realized by new bone formation within the distraction gap. The mechanisms that underlie the DO-induced bone regeneration remain poorly understood and a role of macrophages in the process has been inadequately studied. Here, in a mouse model of DO, we showed significant increase in macrophages in the regeneration area. Moreover, in a loss-of-function approach by depleting inflammatory macrophages, the bone regeneration was compromised by assessment of histology and molecular biology. Thus, our study demonstrates the necessary participation of inflammatory macrophages in the process of DO-induced bone regeneration, and suggests that targeting inflammatory macrophages may help to improve clinical bone repair.