Purpose: Itraconazole is a triazole antifungal drug that has recently been found to inhibit angiogenesis. Itraconazole is a relatively well-tolerated drug but shows hepatotoxicity in a small subset of patients. Itraconazole contains three chiral centers and the commercial itraconazole is composed of four cis-stereoisomers (named IT-A, IT-B, IT-C, and IT-D). We sought to determine whether the stereoisomers of itraconazole might differ in their antiangiogenic activity and hepatotoxicity. Experimental Design: We assessed in vitro antiangiogenic activity of itraconazole and each stereoisomer using human umbilical vein endothelial cell (HUVEC) proliferation and tube formation assays. We also determined their hepatotoxicity using primary human hepatocytes in vitro and a mouse model in vivo. Mouse Matrigel plug and tumor xenograft models were used to evaluate in vivo antiangiogenic and antitumor activities of the stereoisomers. Results: Of the four stereoisomers contained in commercial itraconazole, we found that IT-A (2S,4R,20R) and IT-C (2S,4R,20S) were more potent for inhibition of angiogenesis than IT-B (2R,4S,20R) and IT-D (2R,4S,20S). Interestingly, IT-A and IT-B were more hepatotoxic than IT-C and IT-D. In mouse models, IT-C showed more potent antiangiogenic/antitumor activity with lower hepatotoxicity compared with itraconazole and IT-A. Conclusions: These results demonstrate the segregation of influence of stereochemistry at different positions of itraconazole on its antiangiogenic activity and hepatotoxicity, with the 2 and 4 positions affecting the former and the 20 position affecting the latter. They also suggest that IT-C may be superior to the racemic mixture of itraconazole as an anticancer drug candidate due to its lower hepatotoxicity and improved antiangiogenic activity.