The overexpression of EGFR and/or ErbB2 occurs frequently in ovarian cancers and is associated with poor prognosis. The purpose of this study was to examine the anticancer effects and molecular mechanisms of berberine on human ovarian cancer cells with different levels of EGFR and/or ErbB2. We found that berberine reduced the motility and invasiveness of ovarian cancer cells. Berberine depleted both EGFR and ErbB2 in ovarian cancer cells. Furthermore, berberine suppressed the activation of the EGFR and ErbB2 downstream targets cyclin D1, MMPs, and VEGF by down‐regulating the EGFR‐ErbB2/PI3K/Akt signaling pathway. The berberine‐mediated inhibition of MMP‐2 and MMP‐9 activity could be rescued by co‐treatment with EGF. Finally, we demonstrated that berberine induced ErbB2 depletion through ubiquitin‐mediated proteasome degradation. In conclusion, the suppressive effects of berberine on the ovarian cancer cells that differ in the expression of EGFR and ErbB2 may be mediated by the dual depletion of EGFR and/or ErbB2.