Connexin 43 (Cx43) may be important in cell death and survival due to cell-to-cell communication-independent mechanisms. In our previous study, we found that small G protein signaling modulator 3 (SGSM3), a partner of Cx43, contributes to myocardial infarction (MI) in rat hearts. Based on these previous results, we hypothesized that SGSM3 could also play a role in bone marrow-derived rat mesenchymal stem cells (MSCs), which differentiate into cardiomyocytes and/or cells with comparable phenotypes under low oxygen conditions. Cx43 and Cx43-related factor expression profiles were compared between normoxic and hypoxic conditions according to exposure time, and Sgsm3 gene knockdown (KD) using siRNA transfection was performed to validate the interaction between SGSM3 and Cx43 and to determine the roles of SGSM3 in rat MSCs. We identified that SGSM3 interacts with Cx43 in MSCs under different oxygen conditions and that Sgsm3 knockdown inhibits apoptosis and cardiomyocyte differentiation under hypoxic stress. SGSM3/Sgsm3 probably has an effect on MSC survival and thus therapeutic potential in diseased hearts, but SGSM3 may worsen the development of MSC-based therapeutic approaches in regenerative medicine. This study was performed to help us better understand the mechanisms involved in the therapeutic efficacy of MSCs, as well as provide data that could be used pharmacologically.