Withaferin A (WA), a naturally occurring steroidal lactone, directly binds to Hsp90 and leads to the degradation of Hsp90 client protein. The purpose of this study is to investigate the structure activity relationship (SAR) of withanolides for their inhibition of Hsp90 and anti-proliferative activities in pancreatic cancer cells. In pancreatic cancer Panc-1 cells, withaferin A (WA) and its four analogues withanolide E (WE), 4-hydroxywithanolide E (HWE), 3-aziridinylwithaferin A (AzWA) inhibited cell proliferation with IC50 ranged from 1.0 to 2.8 μM. WA, WE, HWE, and AzWA also induced caspase-3 activity by 21-, 6-, 11- and 15-fold, respectively, in Panc-1 cells, while withaperuvin (WP) did not show any activity. Our data showed that WA, WE, HWE, and AzWA, but not WP, all directly bound to Hsp90 and induced Hsp90 aggregation,hence inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins Akt and Cdk4 through proteasome-dependent pathway in pancreatic cancer cells. However, only WA, HWE and AzWA disrupted Hsp90-Cdc37 complexes but not WE and WP. SAR study suggested that the C-5(6)-epoxy functional group contributes considerably for withanolide to bind to Hsp90, inhibit Hsp90 chaperone activity, and result in Hsp90 client protein depletion. Meanwhile, the hydroxyl group at C-4 of ring A may enhance withanolide to inhibit Hsp90 activity and disrupt Hsp90-Cdc37 interaction. These SAR data provide possible mechanisms of anti-proliferative action of withanolides.