Mitochondria form a dynamic network governed by a balance between opposing fission and fusion processes. Because excessive mitochondrial fission correlates with numerous pathologies, including neurodegeneration, the mechanism governing fission has become an attractive therapeutic strategy. However, targeting fission is a double-edged sword as physiological fission is necessary for mitochondrial function. Fission is trigged by Drp1 anchoring to adaptors tethered to the outer mitochondrial membrane. We designed peptide P259 that distinguishes physiological from pathological fission by specifically inhibiting Drp1′s interaction with the Mff adaptor. Treatment of cells with P259 elongated mitochondria and disrupted mitochondrial function and motility. Sustained in vivo treatment caused a decline in ATP levels and altered mitochondrial structure in the brain, resulting in behavioral deficits in wild-type mice and a shorter lifespan in a mouse model of Huntington’s disease. Therefore, the Mff-Drp1 interaction is critical for physiological mitochondrial fission, motility, and function in vitro and in vivo. Tools, such as P259, that differentiate physiological from pathological fission will enable the examination of context-dependent roles of Drp1 and the suitability of mitochondrial fission as a target for drug development.