Beclin 1, an essential autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members. The dissociation of Beclin 1 from the Bcl-2 inhibitors is essential for its autophagic activity, and therefore is tightly controlled. We recently revealed a novel phosphorylation-based mechanism by which death-associated protein kinase (DAPk) regulates this process. We found that DAPk phosphorylates Beclin 1 on T119, a critical residue within its BH3 domain, and thus promotes Beclin 1 dissociation from Bcl-X(L) and autophagy induction. Here we report that T119 phosphorylation also reduces the interaction between Beclin 1 and Bcl-2, in line with the high degree of structural homology between the BH3 binding pockets of Bcl-2 and Bcl-X(L) proteins. Our results reveal a new phosphorylation-based mechanism that reduces the interaction of Beclin 1 with its inhibitors to activate the autophagic machinery.