Members of the evolutionarily conserved dynamin-related GTPase family mediate numerous cellular membrane remodeling events. Dynamin family functions include the scission of clathrin-coated pits from the plasma membrane, mitochondrial fission, and chloroplast division. Here we report that the divergent eukaryote Trypanosoma brucei possesses a single dynamin family gene, which we have designated TbDLP. Furthermore, a single dynamin family gene is also found in the Leishmania major and Trypanosoma vivax genomes, indicating that this is a conserved feature among the kinetoplastida. TbDLP is most homologous to the DMN/DRP family of dynamin-like proteins. Indirect immunofluorescence microscopy reveals that TbDLP is distributed in punctate structures within the cell that partially co-localize with the mitochondrion when labeled with MitoTracker. To define TbDLP function, we have used RNA interference to silence the TbDLP gene. Reduction of TbDLP protein levels causes a profound alteration in mitochondrial morphology without affecting the structure of other membrane-bound compartments, including the endocytic and exocytic apparatus. The mitochondrial profiles present in wild type trypanosomes fuse and collapse in the mutant cells, and by electron microscopy the mitochondria are found to contain an accumulation of constriction sites. These findings demonstrate TbDLP functions in division of the mitochondrial membrane. Most significantly, as TbDLP is the sole member of the dynamin family in this organism, scission of clathrin-coated pits involved in protein trafficking through the highly active endocytic system in trypanosomes must function in the absence of dynamin. The evolutionary implications of these findings are discussed.