Mitochondrial fission is a highly regulated process mediated by a defined set of protein factors and is involved in the early stage of apoptosis. In mammals, at least two proteins, the dynamin-like protein DLP1/Drp1 and the mitochondrial outer membrane protein hFis1, participate in mitochondrial fission. The cytosolic domain of hFis1 contains six alpha-helices that form two tetratricopeptide repeat (TPR) motifs. Overexpression of hFis1 induces DLP1-mediated fragmentation of mitochondria, suggesting that hFis1 is a limiting factor in mitochondrial fission by recruiting cytosolic DLP1. In the present study, we identified two regions of hFis1 that are necessary for correct fission of mitochondria. We found that the TPR region of hFis1 participates in the interaction with DLP1 or DLP1-containing complex and that the first helix (alpha1) of hFis1 is required for mitochondrial fission presumably by regulating DLP1-hFis1 interaction. Misregulated interaction between DLP1 and hFis1 by alpha1 deletion induced mitochondrial swelling, in part by the mitochondrial permeability transition, but significantly delayed cell death. Our data suggest that hFis1 is a main regulator of mitochondrial fission, controlling the recruitment and assembly of DLP1 during both normal and apoptotic fission processes.