We previously found that 1α, 25‐dihydroxyvitamin D₃ [1α, 25‐(OH)₂‐D₃] modulates adipocyte lipid metabolism via a Ca²⁺‐dependent mechanism and inhibits adipocyte UCP2 expression, indicating that the anti‐obesity effects of dietary calcium are mediated by suppression of 1α, 25‐(OH)₂‐D₃ levels. However, because UCP2 reduces mitochondrial potential, we have evaluated the roles of UCP2, mitochondrial uncoupling, and 1α, 25‐(OH)₂‐D₃ in adipocyte apoptosis. Overexpressing UCP2 in 3T3‐L1 cells induced marked reductions in mitochondrial potential (∆ψ) and ATP production (P<0.01), increases in the expression of caspases (P<0.05), and a decrease in Bcl‐2/Bax expression ratio (P<0.01). Physiological doses of 1α, 25‐(OH)₂‐D₃ (0.1–10 nM) restored mitochondrial ∆ψ in LI‐UCP2 cells and protected against UCP2 overexpression‐induced apoptosis (P<0.01), whereas a high dose (100 nM) stimulated apoptosis in 3T3‐L1 and L1‐UCP2 cells (P<0.05). 1α, 25‐(OH)₂‐D₃ stimulated cytosolic Ca²⁺ dose‐dependently in both 3T3‐L1 and L1‐UCP2 cells. However, physiological doses suppressed mitochondrial Ca²⁺ levels by ~50% whereas the high dose increased mitochondrial Ca²⁺ by 25% (P<0.05); this explains stimulation of apoptosis by the high dose of 1α, 25‐(OH)₂‐D₃. Using high‐calcium diets to suppress 1α, 25‐(OH)₂‐D₃ stimulated adipose tissue apoptosis in aP2 transgenic mice (P<0.01), suggesting that increasing dietary calcium stimulates adipose apoptosis and thereby further contributes to an anti‐obesity effect of dietary calcium.