Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared to compound H-89 against HCT-116 colon cancer cells. The best results were obtained with Compounds a46 and a48 having IC50 values (for HCT-116) of 11.1 and 9.5 µM, respectively, and selectivity indices (IC50 for HEK-293/IC50 for HCT-116) of 12.5 and 16.1, respectively. Through structure-based virtual screening and biological evaluations, we have successfully identified several new Akt inhibitors that displayed cytotoxic activity against HCT-116 human colon cancer cells. Especially, Compounds a46 and a48 may serve as useful lead compounds for further development of new anticancer agents.