Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90 and induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound.