Analgesics and antineoplastic drugs are often used concurrently for cancer patients. Our previous study reported that gap junctions composed of connexin32 (Cx32) was implicated in the effect of analgesics on cisplatin cytotoxicity. However, the effect of analgesic on the most widely expressed connexin (Cx), connexin43 (Cx43), and whether such effect mediates the influence on chemotherapeutic efficiency remain unknown. By manipulation of Cx43 expression or gap junction function, we found that there were gap junction-dependent and independent effect of Cx43 on temozolomide (TMZ) sensitivity in U87 glioblastoma cells. Studies on survival and apoptosis showed widely used analgesic tramadol significantly reduced TMZ-induced cytotoxicity in control and negative control cells but not shCx43-transfected cells. Proliferation assay demonstrated tramadol suppressed TMZ-induced cytotoxicity only on high density (with gap junction formation) but not on low density (without gap junction formation). Tramadol inhibited dye-coupling through gap junctions between U87 cells. Tramadol treatment for 72 h did not alter Cx43 expression, but decreased Cx43 phosphorylation accompanied with reduced p-ERK and p-JNK. Our results indicated that long-term treatment with tramadol reduced TMZ cytotoxicity in U87 cells by suppressing Cx43-composed gap junctions, suggesting identification and usage of antinociceptive drugs which do not downregulate connexin activity should have beneficial therapeutic consequences.