BCL-2 is a negative regulator of apoptosis implicated in homeostatic and pathologic cell survival. The canonical anti-apoptotic mechanism involves entrapment of activated BAX by a groove on BCL-2, preventing BAX homo-oligomerization and mitochondrial membrane poration. The BCL-2 BH4 domain also confers anti-apoptotic functionality, but the mechanism is unknown. We find that a synthetic α-helical BH4 domain binds to BAX with nanomolar affinity and independently inhibits the conformational activation of BAX. Hydrogen-deuterium exchange mass spectrometry demonstrated that the N-terminal conformational changes in BAX induced by a triggering BIM BH3 helix were suppressed by the BCL-2 BH4 helix. Structural analyses localized the BH4 interaction site to a groove formed by residues of α1, α1–α2 loop, and α2–α3 and α5–α6 hairpins on the BAX surface. These data reveal a previously unappreciated binding site for targeted inhibition of BAX and suggest that the BCL-2 BH4 domain may participate in apoptosis blockade by a noncanonical interaction mechanism. Display omitted •Stapled BCL-2 BH4 domain helices directly bind to and inhibit pro-apoptotic BAX•BCL-2 BH4 SAHBs block the conformational activation of BH3-triggered BAX•BCL-2 BH4 domain helices inhibit BAX at a novel interaction site•The BH4-in-groove interaction represents a distinct mechanism for BAX blockade The established paradigm for BCL-2 suppression of apoptosis involves BAX entrapment by a BH3-in-groove mechanism. Barclay et al. discover that a stapled BH4 domain helix of BCL-2 inhibits the conformational activation of BAX by engaging a previously uncharacterized interaction site, revealing a distinct mechanism for BAX blockade and apoptosis modulation.