Mesenchymal stem cells (MSCs) have been increasingly offered for tissue regeneration with the premise that they can survive and thrive amidst the microenvironment of injured or degenerate tissues. The role of high mobility group box 1 (HMGB1) and hypoxia in the proliferation and migration of rat bone marrow MSCs (rBM-MSCs) has been investigated. First, the effect of HMGB1 on the proliferation of rBM-MSCs was determined. Second, to evaluate the regulation of hypoxia and HMGB1 in the migration of rBM-MSCs, cells in the wound healing model were exposed to four conditions: normoxia (20% O2) and complete medium, normoxia and HMGB1, hypoxia (1% O2) and complete medium, hypoxia and HMGB1. RT-PCR and Western blotting were used to measure the expression of migration-related genes and proteins. HMGB1 inhibited the proliferation of rBM-MSCs; HMGB1 alone or together with hypoxia and promoted the migration of MSCs and upregulated the expression of HIF-1α and SDF-1. These results demonstrated that HMGB1 arrested the proliferation of rBM-MSCs, but enhanced the migration of rBM-MSCs which could be further improved by hypoxia. This study strengthens current understanding of the interaction between MSCs and the microenvironment of damaged tissues.