Apoptosis is a critical component of host defense systems for eliminating invading pathogens. Certain viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins that counter host defence responses. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologs in related pox viruses. F1L unexpectedly adopts a novel dimeric Bcl-2-like fold, but no structural data on the mode of engagement of F1L with BH3-domain bearing molecules is available. We now report a crystal structure of MVA F1L in complex with a biochemically identified BH3-domain ligands. Our structure indicate that F1L engages two BH3 ligands at the same time via canonical binding groves, analogous to the manner in which myxoma virus M11L and mammalian anti-apoptotic Bcl-x_L bind their respective ligands. Using structure guided mutagenesis we generated vaccinia virus bearing F1L point mutants that selectively targeted subsets of F1L BH3 domain ligands, and identified Bim as the critical pro-apoptotic effector molecule during vaccinia virus infection.