Objectives: Peroxisome proliferator-activated receptors (PPARs) are implicated in epithelial cell proliferation and differentiation, but investigation has been confounded by potential off-target effects of some synthetic PPAR ligands. Our aim was to determine mechanisms underlying the pro-apoptotic effect of synthetic PPAR agonists in normal human bladder uro-epithelial (urothelial) cells and to reconcile this with the role of PPARs in urothelial cytodifferentiation. Materials and methods: Normal human urothelial (NHU) cells were grown as non-immortal lines in vitro and exposed to structurally diverse agonists ciglitazone, troglitazone, rosiglitazone (PPARγ), ragaglitazar (PPARα/γ), fenofibrate (PPARα) and L165041 (PPARβ/δ). Results: NHU cells underwent apoptosis following acute exposure to ciglitazone, troglitazone or ragaglitazar, but not fenofibrate, L165041 or rosiglitazone, and this was independent of ERK or p38 MAP-kinase activation. Pro-apoptotic agonists induced sustained increases in intracellular calcium, whereas removal of extracellular calcium altered the kinetics of ciglitazone-mediated calcium release from sustained to transient. Cell death was accompanied by plasma-membrane disruption, loss of mitochondrial membrane-potential and caspase-9/caspase-3 activation. PPARγ-mediated apoptosis was unaffected following pre-treatment with PPARγ antagonist T0070907 and was strongly attenuated by store-operated calcium channel (SOC) inhibitors 2-APB and SKF-96365. Conclusions: Our results provide a mechanistic basis for the ability of some PPAR agonists to induce death in NHU cells and demonstrate that apoptosis is mediated via PPAR-independent mechanisms, involving intracellular calcium changes, activation of SOCs and induction of the mitochondrial apoptotic pathway.