Induction of apoptotic cell death is emerging as a promising strategy for prevention and treatment of obesity because removing of adipocytes via apoptosis may result in reducing body fat and a long-lasting maintenance of weight loss. However, the mechanisms controlling adipocyte apoptosis are unknown and even the ability of adipocytes to undergo apoptosis has not been conclusively demonstrated. We have shown previously that the specific Ca(2+) signal, sustained increase in intracellular Ca(2+), triggers apoptotic cell death via activation of Ca(2+)-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is mediated through Ca(2+) signaling. Here, we report that 1,25(OH)(2)D(3) induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca(2+)-dependent calpain and Ca(2+)/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)(2)D(3) induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca(2+). The increase in Ca(2+) was associated with induction of apoptosis and activation of mu-calpain and caspase-12. Our results demonstrate that Ca(2+)-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)(2)D(3) in these cells are Ca(2+)-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.