Heat shock protein 90 (Hsp90) regulates diverse signaling pathways. Emerging evidence suggests that Hsp90 inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), enhance DNA damage-induced cell death, suggesting that Hsp90 may regulate cellular responses to genotoxic stress. However, the underlying mechanisms are poorly understood. Here, we show that the Fanconi anemia (FA) pathway is involved in the Hsp90-mediated regulation of genotoxic stress response. In the FA pathway, assembly of 8 FA proteins including FANCA into a nuclear multiprotein complex, and the complex-dependent activation of FANCD2 are critical events for cellular tolerance against DNA cross-linkers. Hsp90 associates with FANCA, in vivo and in vitro, in a 17-AAG-sensitive manner. Disruption of the FANCA/Hsp90 association by cellular treatment with 17-AAG induces rapid proteasomal degradation and cytoplasmic relocalization of FANCA, leading to impaired activation of FANCD2. Furthermore, 17-AAG promotes DNA cross-linker-induced cytotoxicity, but this effect is much less pronounced in FA pathway-defective cells. Notably, 17-AAG enhances DNA cross-linker-induced chromosome aberrations. In conclusion, our results identify FANCA as a novel client of Hsp90, suggesting that Hsp90 promotes activation of the FA pathway through regulation of intracellular turnover and trafficking of FANCA, which is critical for cellular tolerance against genotoxic stress.