Aim: Impairment of beta-cell function and viability by oxidative stress plays a crucial role during development of type 2 diabetes. We have previously shown that K_ATP channel inhibition by sulfonylureas as well as knockout of the SUR1 subunit attenuate ROS-induced apoptosis via up-regulation of antioxidant enzymes. The present study is intended to elucidate the role of intracellular Ca2+ for sulfonylurea-mediated cell protection. Methods: Apoptosis was determined via active caspase3. Catalase (Cat) and superoxide dismutase (SOD) activities were quantified by suited enzyme assays. Results: Inhibition of K_ATP channels by tolbutamide or gliclazide rendered pancreatic islet cells less sensitive to H2O2-induced apoptotic cell death and up-regulated SOD and Cat activity. To test whether enzyme activation depends on intracellular Ca2+, islets were incubated with gliclazide alone (25 mM, 4h) or in combination with the L-type Ca2+ channel blocker D600 (100 mM). Blocking Ca2+ influx clearly reduced the activating effect of gliclazide on SOD (13±2 vs. 8±1 mU/mg protein, n=4, P<0.05). Determination of caspase3-positive islet cells revealed that protection against H2O2-induced cell death was completely prevented in the presence of D600 (10 mM H2O2: 27±1% apoptotic islet cells, pre-incubation with gliclazide: 14±1%, pre-incubation with gliclazide+D600: 29±1%, n=3, P<0.001 vs. gliclazide alone, ns vs. H2O2 without gliclazide). Interestingly, the beneficial effects of gliclazide on SOD and cell viability were also abolished when Ca2+ sequestration into the endoplasmic reticulum was prevented by treating the islet cells with the SERCA-inhibitor thapsigargin (n=3). Conclusion: Inhibition of K_ATP channels is a useful strategy to elevate the antioxidant capacity of pancreatic islets. The data show that the up-regulation of antioxidant enzymes and protection against ROS-induced apoptosis critically depend on intracellular Ca2+ homeostasis and require intact Ca2+ storage in intracellular compartments.