Calcium store depletion causes opening of the "store operated Ca²⁺ channel" and Ca²⁺ influx [Putney, Cell Calcium 1986], which leads to inhibition of protein tyrosine phosphatase 1B (PTP1B) activity in HEK 293 cells [Sternfeld et al., Cellular Signalling 2005]. Since Ca²⁺ does not inhibit PTP1B directly, we assumed an intermediate signal, which links the rise in cytosolic [Ca²⁺] and PTP1B inhibition. We now show that Ca²⁺ influx is followed by generation of reactive oxygen species (ROS) and that Ca²⁺-dependent inhibition of PTP1B can be abolished in the presence of catalase. Ca²⁺-induced ROS generation is prevented by CCCP (an uncoupler of oxidative phosphorylation) indicating mitochondrial source of ROS. Furthermore, H₂O₂ directly added to cells inhibits PTP1B and leads to increased store depletion-dependent Ca²⁺ influx. PP1, an inhibitor of Src family tyrosine kinases, prevents H₂O₂induced Ca²⁺ influx. We conclude that store depletion-induced Ca²⁺ influx leads to generation of ROS. ROS inhibits PTP1B, which consequently leads to elevated tyrosine phosphorylation of the Ca²⁺ influx channel or of a protein involved in its regulation.