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Thiazolidinediones, which are peroxisome-proliferator-activated receptor γ (PPARγ) agonists that are used as insulin sensitizers for treatment of type 2 diabetes, have been reported to induce apoptosis in cancer cells. The mechanisms by which thiazolidinediones induce apoptosis are undefined. Here, we show that the induction of apoptosis by thiazolidinediones in prostate cancer cell lines is independent of PPARγ activation. Two prostate cancer lines, PC-3 (PPARγ-expressing) and LNCaP (PPARγ-deficient), are both sensitive to apoptosis induction by troglitazone. Also, a troglitazone analogue that lacks binding affinity to PPARγ retained the same ability to induce apoptosis as troglitazone. Moreover, rosiglitazone and pioglitazone, PPARγ agonists with higher binding affinities than troglitazone, do not exhibit correlative apoptotic effects even at high concentration. Target identification studies showed that the apoptotic effect of troglitazone, ciglitazone, and their analogues is in part attributable to their ability to inhibit antiapoptotic protein Bcl-2 and Bcl-xL function. Fluorescence polarization assay indicated that thiazolidinediones disrupt protein-protein interactions between Bak peptide and Bcl-2/Bcl-xL protein at the BH3 domain binding pocket. In addition, co-immunoprecipitation studies showed that treatment of PC-3 cells with troglitazone and its analogue reduced association of Bcl-2 and Bcl-xL with Bak. Transfected LNCaP cells expressing different levels of Bcl-xL were protected from apoptosis after treatment with iii troglitazone and its analogue in a manner that paralleled Bcl-xL expression level. This troglitazone-inducing apoptotic effect involved cytochrome c release and caspase activation. Although troglitazone exhibits Bcl-2/Bcl-xL inhibitory activity, the potency is weak. To improve its potency, three series of analogues are designed and synthesized based on the structure of troglitazone. Among all the analogues synthesized, TG-88 was the best inhibitor of Bcl-2/Bcl-xL function in vitro, and also repressed PC-3 xenograft tumor growth in a nude mouse model.