Dynamin-related proteins (DRPs) can generate forces to remodel membranes. In cells, DRPs require additional proteins [DRP-associated proteins (DAPs)] to conduct their functions. To dissect the mechanistic role of a DAP, we used the yeast mitochondrial division machine as a model, which requires the DRP Dnm1, and two other proteins, Mdv1 and Fis1. Mdv1 played a postmitochondrial targeting role in division by specifically interacting and coassembling with the guanosine triphosphate-bound form of Dnm1. This regulated interaction nucleated and promoted the self-assembly of Dnm1 into helical structures, which drive membrane scission. The nucleation of DRP assembly probably represents a general regulatory strategy for this family of filament-forming proteins, similar to F-actin regulation.