Protein kinase Czeta PKCzeta mediates cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. In the research for its upstream regulator, we investigated the role of Akt2 in chemotaxis and metastasis of human breast cancer cells. Reduction of Akt2 expression by siRNA inhibited chemotaxis of MDA-MB-231, T47D, and MCF7 cells, three representative human breast cancer cells. Expression of a wild type Akt2 in siRNA transfected cells rescued the phenotype. EGF-induced integrin beta1 phosphorylation was dampened, consistent with defects in adhesion. Phosphorylation of LIMK and cofilin, a critical step of cofilin recycle and actin polymerization, was also impaired. Thus, Akt2 regulates both cell adhesion and cytoskeleton rearrangement during chemotaxis. Depletion of Akt2 by siRNA impaired the activation of PKCzeta while inhibition of PKCzeta did not interfere with EGF induced phosphorylation of Akt. Furthermore, EGF induced co-immunoprecipitation between PKCzeta and Akt2, but not Akt1, suggesting that a direct interaction between PKCzeta and Akt2 in chemotaxis. Protein levels of integrin beta1, LIMK, cofilin, and PKCzeta didn't alter, suggesting that Akt2 does not regulate the expression of these signaling molecules. In a Severe Combine Immunodeficiency mouse model, Akt2 depleted MDA-MB-231 cells showed a marked reduction in metastasis to mouse lungs, demonstrating the biological relevancy of Akt2 in cancer metastasis in vivo. Taken together, our results suggest that Akt2 directly mediates EGF-induced chemotactic signaling pathways through PKCzeta and its expression is critical during the extravasation of circulating cancer cells.