The ability of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) systems to kill tumor cells is partially dependent on the integrity of gap junction intercellular communication (GJIC) of targeted tumor cells. Recent studies have suggested that connexin 43 (Cx43), which serves a role in gap junction-mediated intercellular communication, is regulated by small ubiquitin-like modifiers (SUMOs). However, the roles of these post-translational modifications remain to be elucidated. The present study demonstrated overexpression of SUMO‑conjugating enzyme Ubc9 (Ubc9) protein in osteosarcoma. Silencing Ubc9 by siRNA inhibited osteosarcoma cell proliferation and migration, and significantly increased the sensitivity of cells to HSV-TK/GCV systems both in vitro and in vivo. Further experimentation demonstrated that silencing Ubc9 induced decoupling of SUMO1 from Cx43, generating increased free Cx43 levels, which is important for reconstructing GJIC and recovering cellular functions. In conclusion, the present study revealed a novel method for the effective restoration of GJIC in osteosarcoma cells, which may increase their sensitivity to conventional chemotherapy.