Androgen ablation therapy represents the first line of therapeutic intervention in men with advanced or recurrent prostate tumors. However, the incomplete efficacy and lack of durable response to this clinical strategy highlights an urgent need for alternative treatment options to improve patient outcomes. Targeting the molecular chaperone heat shock protein 90 (Hsp90) represents a potential avenue for therapeutic intervention as its inhibition results in the coordinate blockade of multiple oncogenic signaling pathways in cancer cells. Moreover, Hsp90 is essential for the stability and function of numerous client proteins, a number of which have been causally implicated in the pathogenesis of prostate cancer, including the androgen receptor (AR). Here, we examined the preclinical activity of ganetespib, a small molecule inhibitor of Hsp90, in a panel of prostate cancer cell lines. Ganetespib potently decreased viability in all lines, irrespective of their androgen sensitivity or receptor status, and more effectively than the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Interestingly, while ganetespib exposure decreased AR expression and activation, the constitutively active V7 truncated isoform of the receptor was unaffected by Hsp90 inhibition. Mechanistically, ganetespib exerted concomitant effects on mitogenic and survival pathways, as well as direct modulation of cell cycle regulators, to induce growth arrest and apoptosis. Further, ganetespib displayed robust antitumor efficacy in both AR-negative and positive xenografts, including those derived from the 22Rv1 prostate cancer cell line that co-expresses full-length and variant receptors. Together these data suggest that further investigation of ganetespib as a new therapeutic treatment for prostate cancer patients is warranted.