Datasets that support the figures published in Lemos et al., Cells (2023). The project aims to elucidate the role of the metabolic enzyme PKM2 in intracellular Ca2+ signaling. We demonstrated that PKM2 can differentially regulate cytosolic and mitochondrial Ca2+ handling. In the cytosol, PKM2 interacts with and inhibits IP3R activity, while in the mitochondria, PKM2 modulates the Ca2+ signal by influencing mitochondrial metabolism, structure, and dynamics. By using the TAT-D5SD peptide, corresponding to amino acids 2078-2098 of IP3R1, which disrupts the PKM2:IP3R complex, we observed that the effects of this peptide on Ca2+ handling and apoptosis depend on the IP3R but not on PKM2. Furthermore, the presence of PKM1 and the tyrosine kinase Lck is not required, suggesting the presence of one or more other proteins that can interact with the IP3R and form a complex at the same site as PKM2 on the IP3R.