Hepatocellular carcinoma (HCC) is generally believed to have low sensitivity to chemotherapeutic agents including oxaliplatin (OXA). Studies have demonstrated that gap junctions (GJs) composed of connexin (Cx) proteins have the potential to modulate drug chemosensitivity in multiple tumor cells. In the present study, we investigated the characteristics of Cx and GJs in HCC at both histologic and cytologic levels, and the effects of GJ and its effective components on OXA cytotoxicity in HCC cells in vitro. Immunohistochemistry was performed in 76 HCCs and 20 normal liver tissues to detect and locate the expression of Cx26, Cx32 and Cx43. At cytologic levels, the expression and localization of Cxs were evaluated by RT-PCR, western blot and immunofluorescence assay, respectively. The GJ function between adjacent cells was detected using dye transfer assay. The role of GJs in the modulation of OXA toxicity in HCC cells was explored using pharmacologic and molecular biologic methods. We found that Cx expression in HCC tissues was significantly lower than in normal liver tissues, and the 'internalization' from cell membrane to cytoplasm was remarkable. In vitro experiments revealed the presence of functional GJs in the SMMC-7721 HCC cells due to a small amount of Cx protein along the plasma membrane at cell-cell contacts. Regulation of this part of GJs positively influenced OXA cytotoxicity. Using RNA interference, only specific inhibition of Cx26 but not Cx32 or Cx43 reduced OXA cytotoxicity. Conversely, Cx26 overexpression by transfection of Cx26 plasmid DNA enhanced OXA cytotoxicity. This study demonstrated that during hepatocarcinogenesis, the reduced expression and internalization of Cx proteins impaired the GJ function, which further attenuated OXA cytotoxicity. Impaired GJ function may contribute to low intrinsic chemosensitivity of HCC cells to OXA, mediated by Cx26.