Tamoxifen is a triphenylethylene non-steroidal antiestrogen anticancer agent. It also shows inhibitory effects on metastasis of estrogen receptor (EsR)-independent tumors, but the underlying mechanism is unclear. It was demonstrated in this study that, in EsR-negative and highly metastatic human hepatocellular carcinoma MHCC97H cells, tamoxifen-inhibited cell migration, volume-activated Cl currents (ICl,vol) and regulatory volume decrease (RVD) in a concentration-dependent manner with a similar IC50. Analysis of the relationships between migration, ICl,vol and RVD showed that cell migration was positively correlated with ICl,vol and RVD. Knockdown of the expression of ClC-3 Cl channel proteins by ClC-3 shRNA or siRNA inhibited ICl,vol, and cell migration, and these inhibitory effects could not be increased further by addition of tamoxifen in the medium. The results suggest that knockdown of ClC-3 expression may deplete the effects of tamoxifen; tamoxifen may inhibit cell migration by modulating ICl,vol and cell volume. Moreover, tamoxifen decreased the activity of protein kinase C (PKC) and the effects were reversed by the PKC activator PMA. Activation of PKC by PMA could competitively downregulate the inhibitory effects of tamoxifen on ICl,vol. PMA promoted cell migration, and knockdown of ClC-3 expression by ClC-3 siRNA abolished the PMA effect on cell migration. The results suggest that tamoxifen may inhibit ICl,vol by suppressing PKC activation; ICl,vol may be an EsR-independent target for tamoxifen in the anti-metastatic action on cancers, especially on EsR-negative cancers. The finding may have an implication in the clinical use of tamoxifen in the treatments of both EsR-positive and EsR-negative cancers. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.