The study explores the functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1, revealing a novel autophagy-stimulatory role for BH3-only proteins beyond their established role in apoptosis. Beclin-1, an essential autophagy mediator, interacts with Bcl-2 and Bcl-XL through a BH3-like domain (residues 114–123). This interaction is disrupted by mutations in either the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-XL, leading to the release of Beclin-1 from inhibition by Bcl-2/Bcl-XL and subsequent autophagy stimulation. The BH3 mimetic ABT737 competitively inhibits the Beclin-1-Bcl-2/Bcl-XL interaction, antagonizes autophagy inhibition, and stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, while overexpression of Bad induces autophagy. Similarly, gain-of-function mutation of the sole BH3-only protein EGL-1 in Caenorhabditis elegans induces autophagy, while its deletion compromises starvation-induced autophagy. These findings demonstrate that BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the Beclin-1-Bcl-2/Bcl-XL interaction. The study also highlights the role of BH3-only proteins in regulating autophagy, with implications for cancer therapy using BH3 mimetics to sensitize tumor cells to apoptosis. The results suggest a novel regulatory mechanism involving BH3-only proteins in autophagy, with potential therapeutic applications in cancer treatment.The study explores the functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1, revealing a novel autophagy-stimulatory role for BH3-only proteins beyond their established role in apoptosis. Beclin-1, an essential autophagy mediator, interacts with Bcl-2 and Bcl-XL through a BH3-like domain (residues 114–123). This interaction is disrupted by mutations in either the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-XL, leading to the release of Beclin-1 from inhibition by Bcl-2/Bcl-XL and subsequent autophagy stimulation. The BH3 mimetic ABT737 competitively inhibits the Beclin-1-Bcl-2/Bcl-XL interaction, antagonizes autophagy inhibition, and stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, while overexpression of Bad induces autophagy. Similarly, gain-of-function mutation of the sole BH3-only protein EGL-1 in Caenorhabditis elegans induces autophagy, while its deletion compromises starvation-induced autophagy. These findings demonstrate that BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the Beclin-1-Bcl-2/Bcl-XL interaction. The study also highlights the role of BH3-only proteins in regulating autophagy, with implications for cancer therapy using BH3 mimetics to sensitize tumor cells to apoptosis. The results suggest a novel regulatory mechanism involving BH3-only proteins in autophagy, with potential therapeutic applications in cancer treatment.