Mitochondria are dynamic organelles that undergo regulated fission and fusion events that are essential to maintain metabolic stability. We previously demonstrated that the mitochondrial fission GTPase DRP1 is a substrate for SUMOylation. To further understand how SUMOylation impacts mitochondrial function, we searched for a SUMO protease that may affect mitochondrial dynamics. We demonstrate that the cytosolic pool of SENP5 catalyzes the cleavage of SUMO1 from a number of mitochondrial substrates. Overexpression of SENP5 rescues SUMO1-induced mitochondrial fragmentation that is partly due to the downregulation of DRP1. By contrast, silencing of SENP5 results in a fragmented and altered morphology. DRP1 was stably mono-SUMOylated in these cells, suggesting that SUMOylation leads to increased DRP1 mediated fission. In addition, the reduction of SENP5 levels resulted in a significant increase in the production of free radicals. Reformation of the mitochondrial tubules by expressing the dominant interfering DRP1 or by RNA silencing of endogenous DRP1 protein rescued both the morphological aberrations and the increased production of ROS induced by downregulation of SENP5. These data demonstrate the importance of SENP5 as a new regulator of SUMO1 proteolysis from mitochondrial targets, impacting mitochondrial morphology and metabolism.