Fluoxetine, a widely used anti-depressant compound, has several additional effects, including blockade of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells by using fura-2-based digital calcium imaging and assay for [3H]-inositol phosphates (IPs). Treatment with ATP (100μM) for 2 min induced [Ca2⁢]i increases. The ATP-induced [Ca2⁢]i increases were significantly decreased by removal of extracellular Ca2⁢ and treatment with the inhibitor of endoplasmic reticulum Ca2⁢ ATPase thapsigargin (1μM). Treatment with fluoxetine for 5 min blocked the ATP-induced [Ca2⁢]i increase concentration-dependently. Treatment with fluoxetine (30μM) for 5 min blocked the ATP-induced [Ca2⁢]i increase following removal of extracellular Ca2⁢ and depletion of intracellular Ca2⁢ stores. While treatment with the L-type Ca2⁢ channel antagonist nimodipine for 10 min inhibited the ATP-induced [Ca2⁢]i increases significantly, treatment with fluoxetine alone blocked the ATP-induced responses. Treatment with fluoxetine also inhibited the 50 mM K⁢-induced [Ca2⁢]i increases completely. However, treatment with fluoxetine did not inhibit the ATP-induced [3H]-IPs formation. Collectively, we conclude that fluoxetine inhibits ATP-induced [Ca2⁢]i increases in PC12 cells by inhibiting both an influx of extracellular Ca2⁢ and a release of Ca2⁢ from intracellular stores without affecting IPs formation.