Glioblastoma is the most common and aggressive brain tumor and it is characterized by a high mortality rate. Temozolomide (TMZ) is an effective chemotherapy drug for glioblastoma, but the resistance to TMZ has come to represent a major clinical problem, and its underlying mechanism has yet to be elucidated. In the present study, the role of exosomal connexin 43 (Cx43) in the resistance of glioma cells to TMZ and cell migration was investigated. First, higher expression levels of Cx43 were detected in TMZ‑resistant U251 (U251r) cells compared with those in TMZ‑sensitive (U251s) cells. Exosomes from U251s or U251r cells (sExo and rExo, respectively) were isolated. It was found that the expression of Cx43 in rExo was notably higher compared with that in sExo, whereas treatment with rExo increased the expression of Cx43 in U251s cells. Additionally, exosomes stained with dioctadecyloxacarbocyanine (Dio) were used to visualized exosome uptake by glioma cells. It was observed that the uptake of Dio‑stained rExo in U251s cells was more prominent compared with that of Dio‑stained sExo, while Gap27, a gap junction mimetic peptide directed against Cx43, alleviated the rExo uptake by cells. Moreover, rExo increased the IC of U251s to TMZ, colony formation and Bcl‑2 expression, but decreased Bax and cleaved caspase‑3 expression in U251s cells. Gap27 efficiently inhibited these effects of rExo on U251s cells. Finally, the results of the wound healing and Transwell assays revealed that rExo significantly enhanced the migration of U251s cells, whereas Gap27 significantly attenuated rExo‑induced cell migration. Taken together, these results indicate the crucial role of exosomal Cx43 in chemotherapy resistance and migration of glioma cells, and suggest that Cx43 may hold promise as a therapeutic target for glioblastoma in the future.