AbstractNogo is a potent inhibitor of regeneration following spinal cord injury. To develop a better understanding of the mechanisms responsible for regenerative failure we used a yeast two‐ hybrid approach to try and identify proteins that interact with Nogo. We identified a novel mitochondrial protein designated Nogo‐interacting mitochondrial protein (NIMP) in a screen of an adult human brain cDNA library. This interaction was confirmed by co‐immunoprecipitation in both brain tissue (endogenous) and transfected HEK293T cells (overexpressed). In support of these studies we demonstrate that Nogo interacts with the UQCRC1 and UQCRC2 components of complex III, within the mitochondrial respiratory chain. The mitochondrial localization of NIMP was evidenced by confocal image analysis and western blot analysis of isolated mitochondria. NIMP is highly conserved and ubiquitously expressed in mitochondria‐enriched tissues. Within the CNS, NIMP‐like immunoreactivity is present in neurons and astrocytes. These data suggest that NIMP is a novel mitochondrial protein that interacts with Nogo. The interaction of Nogo with mitochondrial proteins may provide insight into the mechanisms for Nogo‐induced inhibition of neurite growth.