Chemotherapy is one of the most commonly used therapeutic strategies for metastatic colon cancer. However, the development of resistance to chemotherapeutic agents limits their application in clinical use. The underlying mechanisms of this resistance development require further elucidation. The current study investigated the effects of connexin43 (Cx43) gap junctions on 5‑fluorouracil (5‑FU), oxaliplatin and irinotecan in colon cancer cells. Three different methods were used to manipulate Cx43 gap junction function: i) Cell culture at different densities; ii) pretreatment with a Cx43 specific inhibitor or enhancer; and iii) Cx43 gene knock‑down. Results indicated that the cell toxicity of 5‑FU, oxaliplatin and irinotecan was cell density‑dependent, which was mediated by gap junctions. Downregulation of Cx43 gap junction functioning attenuated 5‑FU, oxaliplatin and irinotecan toxicity in colon cancer cells, which was increased in cells treated with a Cx43 gap junction function enhancer. Thus, the results of the present study suggest that resistance to 5‑FU, oxaliplatin and irinotecan in colon cancer cells was relative to Cx43 expression loss as cancer developed, which may indicate a novel basis for therapeutic strategy development to combat drug resistance in numerous cell types, in addition to colon cancer cells.