Succinate dehydrogenase (SDH) occupies a central place in cellular energy production, linking the tricarboxylic cycle with the electron transport chain. As a result, a subset of cancers and neuromuscular disorders result from mutations affecting any of the four SDH structural subunits or either of two known SDH assembly factors. Herein we characterize an evolutionarily conserved SDH assembly factor designated Sdh8/SDHAF4, using yeast, Drosophila, and mammalian cells. Sdh8 interacts specifically with the catalytic Sdh1 subunit in the mitochondrial matrix, facilitating its association with Sdh2 and the subsequent assembly of the SDH holocomplex. These roles for Sdh8 are critical for preventing motility defects and neurodegeneration in Drosophila as well as the excess ROS generated by free Sdh1. These studies provide insights into the mechanisms by which SDH is assembled and raise the possibility that some forms of neuromuscular disease may be associated with mutations that affect this SDH assembly factor.