Certain members of the thiazolidenedione family of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, such as troglitazone and ciglitazone, exhibit antitumor effects; however, the underlying mechanism remains inconclusive. This study shows that the effect of these thiazolidenedione members on apoptosis in prostate cancer cells is independent of PPARgamma activation. First, close structural analogues of thiazolidenediones, whereas devoid of PPARgamma activity, retain the ability to induce apoptosis with equal potency. Second, both PC-3 (PPARgamma-expressing) and LNCaP (PPARgamma-deficient) cells are sensitive to apoptosis induction by troglitazone and its PPARgamma-inactive analogue irrespective of their PPARgamma expression status. Third, rosiglitazone and pioglitazone, potent PPARgamma agonists, show marginal effects on apoptosis even at high concentrations. Evidence indicates that the apoptotic effect of troglitazone, ciglitazone, and their PPARgamma-inactive analogues 5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-benzylidene]-2,4-thiazolidine-dione (Delta2-TG) and 5-[4-(1-methyl-cyclohexylmethoxy)-benzylidene]-thiazolidine-2,4-dione, respectively, is in part attributable to their ability to inhibit the anti-apoptotic functions of Bcl-xL and Bcl-2. Treatment of PC-3 cells with troglitazone or Delta2-TG led to reduced association of Bcl-2 and Bcl-xL with Bak, leading to caspase-dependent apoptosis. Bcl-xL overexpression protects LNCaP cells from apoptosis induction by troglitazone and Delta2-TG in an expression level-dependent manner. Considering the pivotal role of Bcl-xL/Bcl-2 in regulating mitochondrial integrity, this new mode of mechanism provides a framework to account for the PPARgamma-independent action of thiazolidenediones in inducing apoptosis in cancer cells. Moreover, dissociation of these two pharmacologic activities provides a molecular basis to develop novel Bcl-xL/Bcl-2 inhibitors, of which the proof of principle is illustrated by a Delta2-TG analogue with potent in vivo antitumor activities.