This study identifies a novel mechanism by which thiazolidinediones mediate cyclin D1 repression in prostate cancer cells. Based on the finding that the thiazolidinedione family of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists mediated PPARgamma-independent cyclin D1 degradation, we developed a novel PPARgamma-inactive troglitazone derivative, STG28, with high potency in cyclin D1 ablation. STG28-mediated cyclin D1 degradation was preceded by Thr-286 phosphorylation and nuclear export, which however, were independent of glycogen synthase kinase 3beta. Mutational analysis further confirmed the pivotal role of Thr-286 phosphorylation in STG28-induced nuclear export and proteolysis. Of several kinases examined, inhibition of IkappaB kinase alpha blocked STG28-mediated cytoplasmic sequestration and degradation of cyclin D1. Pulldown of ectopically expressed Cul1, the scaffold protein of the Skp-Cullin-F-box E3 ligase, in STG28-treated cells revealed an increased association of cyclin D1 with beta-TrCP, whereas no specific binding was noted with other F-box proteins examined, including Skp2, Fbw7, Fbx4, and Fbxw8. This finding represents the first evidence that cyclin D1 is targeted by beta-TrCP. Moreover, beta-TrCP expression was up-regulated in response to STG28, and ectopic expression and small interfering RNA-mediated knock-down of beta-TrCP enhanced and protected against STG28-facilitated cyclin D1 degradation, respectively. Because cyclin D1 lacks the DSG destruction motif, mutational and modeling analyses indicate that cyclin D1 was targeted by beta-TrCP through an unconventional recognition site, (279)EEVDLACpT(286), reminiscent to that of Wee1. Moreover, we obtained evidence that this beta-TrCP-dependent degradation takes part in controlling cyclin D1 turnover when cancer cells undergo glucose starvation, which endows physiological relevance to this novel mechanism.