Mitochondrial respiratory chain disorders are characterized by loss of electron transport chain (ETC) activity. Although the causes of many such diseases are known, there is a lack of effective therapies. To identify genes that confer resistance to severe ETC dysfunction when inactivated, we performed a genome-wide genetic screen in haploid human cells with the mitochondrial complex III inhibitor antimycin. This screen revealed that loss of ATPIF1 strongly protects against antimycin-induced ETC dysfunction and cell death by allowing for the maintenance of mitochondrial membrane potential. ATPIF1 loss protects against other forms of ETC dysfunction and is even essential for the viability of human rho degrees cells lacking mitochondrial DNA, a system commonly used for studying ETC dysfunction. Importantly, inhibition of ATPIF1 ameliorates complex III blockade in primary hepatocytes, a cell type afflicted in severe mitochondrial disease. Altogether, these results suggest that inhibition of ATPIF1 can ameliorate severe ETC dysfunction in mitochondrial pathology.