We previously reported that connexin (Cx) 26 expression is involved in negative growth control of HepG2 cells established from a human hepatoma. We also found that induction of E-cadherin and subsequent formation of a cell adhesion complex were induced in HepG2 cells by C × 26 expression. To examine the exact role of C × 26-induced E-cadherin junctions in regulating appearance of malignant phenotypes of HepG2 cells, we expressed a C × 26 antisense oligodeoxynucleotide (AS-ODN) in an established HepG2 cell clone that has stable expression of C × 26 genes. We investigated changes in the expression of E-cadherin, the localization of β-catenin, and some malignant phenotypes of HepG2 clone after the suppression of C × 26 expression by AS-ODN treatment. The AS-ODN treatment prevented the expression of C × 26 and E-cadherin, and the localization of β-catenin was changed from cytoplasmic membrane to the cytoplasm. In parallel, a morphological change from a monolayer of polygonal cells to multilayered colonies was induced by the treatment, indicating a change of a malignant phenotype of HepG2 cells. The activity of matrix metalloproteinase 9 (MMP-9) was elevated by the AS-ODN treatment. A concomitant increase in invasiveness of the C × 26-expressing cells by the treatment was also observed in an in vitro assay with Matrigel matrix. These results suggest that the induction of E-cadherin and formation of the cell adhesion complex by C × 26 expression contribute to the reversal of some malignant phenotypes of HepG2 cells. Furthermore, the C × 26-dependent expression of E-cadherin leads to reduction of the invasiveness of the cells through suppression of MMP-9 activity. © 2001 Wiley-Liss, Inc.