Mitochondrial structure can be maintained at steady state or modified in response to changes in cellular physiology. This is achieved by the coordinated regulation of dynamic properties including mitochondrial fusion, division, and transport. Disease states, including neurodegeneration, are associated with defects in these processes. In vertebrates, two mitofusin paralogues, Mfn1 and Mfn2, are required for efficient mitochondrial fusion. The mitofusins share a high degree of homology and have very similar domain architecture, including an amino terminal GTPase domain and two extended helical bundles that are connected by flexible regions. Mfn1 and Mfn2 are nonredundant and are both required for mitochondrial outer membrane fusion. However, the molecular features that make these proteins functionally distinct are poorly defined. By engineering chimeric proteins composed of Mfn1 and Mfn2, we discovered a region that contributes to isoform-specific function (mitofusin isoform-specific region [MISR]). MISR confers unique fusion activity and mitofusin-specific nucleotide-dependent assembly properties. We propose that MISR functions in higher-order oligomerization either directly, as an interaction interface, or indirectly through conformational changes.