Background <p>We have reported that the cytokines and chemokines contained in conditioned medium of human mesenchymal stem cells (MSC-CM), which were derived from bone marrow, promote bone regeneration. We recently reported macrophage phenotype polarization towards the anti-inflammatory M2 phenotype induced by MSC-CM and its potential to establish regenerative condition and assist subsequent bone regeneration. However, the specific factors in the MSC-CM responsible for this process remain unclear. Monocyte chemoattractant protein (MCP) -1, present in MSC-CM, promotes cell migration and activation of the monocyte-macrophage lineage; therefore, we hypothesized that MCP-1 is one of the key factors in MSC-CM-induced macrophage phenotype polarization. The effect of MCP-1 on MSC-CM-induced macrophage phenotype polarization and subsequent bone regeneration was investigated in this study.</p> Methods <p>MCP-1 was depleted from MSC-CM (depMSC-CM) and used in subsequent experiments. Rat bone marrow macrophages were incubated in MSC-CM or depMSC-CM and expression of macrophage markers was examined in vitro. In addition, the effect of MSC-CM and depMSC-CM on bone regeneration and macrophage phenotype polarization were evaluated using rat calvaria defect model in vivo.</p> Results <p>MSC-CM enhanced M2 macrophage marker expression in rat bone marrow macrophages compared to those treated with depMSC-CM in vitro. In addition, MSC-CM increased the number of M2 macrophage marker-positive cells in bone defects and enhanced subsequent bone regeneration in a rat calvaria bone defect model.</p> Conclusions <p>MCP-1 seemed to be a one of the most contributing factors in MSC-CM-induced macrophage phenotypic polarization and subsequent bone regeneration.</p>