Berberine is an active ingredient extracted from Coptidis rhizoma which has been used for centuries as a traditional Chinese medicine for treatment of inflammatory diseases. Recent studies have indicated that berberine has anticancer properties. Berberine arrested cell growth and inhibited cell migration in various cancer cell lines. In this study, we examined the effects of berberine on HONE1 cells, which have been commonly used as a cell model for nasopharyngeal carcinoma. We observed the inhibitory effects of berberine on HONE1 cells at a high dosage (>150 microM). Berberine effectively induced the mitotic arrest of HONE1 cells at 300 microM which was associated with apoptosis. Berberine had differential intracellular localization at low and high doses. At a low dose (50 microM), berberine was localized in the mitochondria while at a high dose (300 microM), berberine was localized in the nucleus which may have induced mitotic arrest. Berberine effectively inhibited cell migration and invasion at low doses. Using a specific GST pull-down assay of activated Rho GTPases, we demonstrated that berberine suppressed the activation of Rho GTPases including RhoA, Cdc42 and Rac1. This indicates a novel function of berberine in the suppression of Rho GTPase signaling to mediate its inhibitory action on cell migration and motility. The potential of berberine to inhibit cancer metastasis in cancer warrants further investigation.