High mobility group box 1 (HMGB1) epxression has been found in the inflammatory microenvironment of fractures. It is well known that HMGB1 acts as a chemoattractant for mesenchymal stem cells (MSCs); however, the effects of HMGB1 on MSC migration and osteoblastic differentiation, and the signaling pathways involved in these effects, have not yet been elucidated. In this study, we aimed to investigate these effects, as well as the signaling mechanisms involved, using in vitro models. We found that HMGB1, in varying concentrations, promoted the osteoblastic differentiation of MSCs, the synthesis of receptor for advanced glycation end products (RAGE) and Toll-like receptor (TLR)2/4, and the activation of the p38 mitogen-activated protein kinase (MAPK) and nuclear factor‑κB (NF‑κB) signaling pathways. Subsequently, we cultured the MSCs in the appropriate concentration of HMGB1, and determined the signaling pathways involved in the effects of HMGB1 on MSC migration and differentiation, using receptor neutralizing antibodies and signaling pathway inhibitors. From the results of this study, we concluded that HMGB1 promotes MSC migration through the activation of the p38 MAPK signaling pathway, and also promotes MSC differentiation by binding to TLR2/4 and activating the p38 MAPK signaling pathway. These findings elucidate the mechanisms underlying the effects of HMGB1 in the fracture microenvironment, which may provide a theoretical basis for the development of improved clinical treatments for fractures.