To better elucidate the role of macrophages in bone morphogenetic protein (BMP)-induced bone repair, this study evaluated the effects of macrophages on the migration, metabolic activity and BMP-2-induced osteogenic differentiation of human mesenchymal stem cells (hMSCs). Human monocytes were induced into a macrophage phenotype, and the conditioned media (CM) from undifferentiated monocytes and differentiated macrophages were collected for treatment of hMSCs. Expression levels of osteoblastic marker genes, alkaline phosphatase (ALP) activity and mineral deposition were assessed. The migration of hMSCs was significantly decreased after treatment with the macrophage CM (but not monocyte CM), in a dose-dependent manner. Significant inhibition of hMSC metabolism was observed on days 3 and 7 after treatment with the macrophage CM. The osteoblastic marker genes analyzed (ALP, bone sialoprotein, osteocalcin and runt-related transcription factor-2) after exposure of hMSCs to BMP-2 were all significantly downregulated in cells treated with the macrophage CM. The hMSCs treated with macrophage CM showed significantly decreased enzymatic activity of ALP and calcium content compared with those treated with monocyte CM or basal medium. High levels of interleukin-1β and tumor necrosis factor-α found in macrophage CM may mediate these observed effects on hMSCs. We conclude that macrophage CM suppressed the BMP-2-induced osteogenic differentiation of hMSCs, suggesting that macrophages might contribute to decreased osteogenic effects of BMPs in a clinical setting.