© 2019, Springer Nature Switzerland AG. Bcl-2 proteins have emerged as critical regulators of intracellular Ca 2+ dynamics by directly targeting and inhibiting the IP 3 receptor (IP 3 R), a major intracellular Ca 2+ -release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP 3 R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP 3 R function provoked by low concentration of agonist or IP 3 , while fails to attenuate against high concentration of agonist or IP 3 . Surprisingly, Bcl-2 remained capable of inhibiting IP 3 R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380–1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP 3 R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP 3 and adenophostin A, ligands of the IP 3 R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP 3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP 3 R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2’s inhibitory properties on IP 3 Rs that is tunable to the level of IP 3 signaling in cells.