SignificanceApoptosis-inducing factor 1 (AIFM1) resides within the intermembrane space of mitochondria and upon programmed cell death was found to induce chromatin condensation and DNA fragmentation. While the apoptosis-related role of AIFM1 is well understood, recent findings pointed to additional, not well-characterized functional roles of AIFM1 in oxidative phosphorylation. Using cross-linking mass spectrometry and complexome profiling, we uncover that a substantial amount of dimeric AIFM1 is engaged with ∼10% of monomeric cytochromecoxidase (COX). Further structural modeling and restraint-driven docking structurally characterize a COX-AIFM1₂complex, not only highlighting how AIFM1 might be N-terminally inserted into the inner mitochondrial membrane but also providing clues on potential functional implications including an involvement in promoting apoptosis.