Reactive oxygen species (ROS) stimulate cytoplasmic Ca2+ (Ca2+c) signaling, but the exact role of the IP3 receptors (IP3R) in this process remains unclear. IP3Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP3Rs at the ER-mitochondrial associations. Also, IP3Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O2⨪) on IP3R-mediated Ca2+ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed Ca2+c spikes and frequency-modulated oscillations evoked by a O2⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The Ca2+c signal was mediated by ER Ca2+ mobilization. X+XO added to permeabilized cells promoted the Ca2+c rise evoked by submaximal doses of IP3, indicating that O2⨪ directly sensitizes IP3R-mediated Ca2+ release. In response to X+XO, DT40 cells lacking two of three IP3R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP3Rs (DKO2) showed a Ca2+c signal, whereas DKO expressing type 3 IP3R (DKO3) did not. By contrast, IgM that stimulates IP3 formation, elicited a Ca2+c signal in every DKO. X+XO also facilitated the Ca2+ release evoked by submaximal IP3 in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP3R (TKO). However, X+XO could also facilitate the effect of suboptimal IP3 in TKO transfected with rat IP3R3. Although in silico studies failed to identify a thiol missing in the chicken IP3R3, an X+XO-induced redox change was documented only in the rat IP3R3. Thus, ROS seem to specifically sensitize IP3Rs through a thiol group(s) within the IP3R, which is probably inaccessible in the chicken IP3R3. Background: Reactive oxygen species (ROS) affect cytoplasmic calcium signaling. Results: Superoxide anion causes oxidation of the IP3 receptor and sensitization of calcium release to promote cytoplasmic calcium oscillations and mitochondrial calcium uptake. Conclusion: Physiologically relevant ROS controls cytoplasmic and mitochondrial calcium transport through IP3 receptors. Significance: Mechanisms of calcium and ROS interactions are relevant for both physiological and pathophysiological signaling.