Although ErbB2 is known to enhance breast cancer metastasis, the signaling events responsible for this remain elusive. a-Isozyme of protein kinase C (PKCa), which is involved in cancer development and progression, has been suggested to be activated by ErbB2 without direct evidence. In addition, the roles of PKCa in ErbB2-mediated cancer cell malignancy have not been clearly identified. In this study, we investigated whether ErbB2 can activate PKCa and determined what role PKCa plays in ErbB2-mediated breast cancer cell invasion. We expressed wild-type and mutant ErbB2 with altered signaling capacities in MDA-MB-435 breast cancer cells and revealed that overexpression or activation of ErbB2 in MDA-MB-435 cells upregulated and activated PKCa and that downregulation of ErbB2 by small-interfering RNA decreased the expression and activity of PKCa in BT474 breast cancer cells. These in vitro results were supported by data from breast cancer patient samples. In 150 breast cancer tumor samples, ErbB2-overexpressing tumors showed significantly higher positive rates of PKCa membrane immunohistochemistry staining than that of ErbB2-low-expressing tumors. Mechanistically, we found that PKCa is co-immunoprecipitated with Src and PKCa expression and activity can be decreased by Src inhibitor PP2 and by the expression of a dominantnegative mutant of Src. Moreover, ErbB2-mediated upregulation of urokinase-type plasminogen activator receptor (uPAR) is reduced by either the PKCa inhibitor Go6976 or the Src inhibitor PP2, and the combination of Go6976 with PP2 is superior to either agent alone in suppressing uPAR expression and cell invasion. These results demonstrate that PKCa is critical for ErbB2-mediated cancer cell invasion and provide valuable insights for current and future PKCa and Src inhibitor clinical trials.