Mitochondrial Rho GTPase (MIRO) is a scaffold protein anchored to the outer mitochondrial membrane, where it regulates mitochondrial trafficking through interactions with the motor adaptor Trafficking Kinesin-binding protein (TRAK). MIRO1 contains N- and C-terminal GTPase domains flanking two pairs of EF-hands. While MIRO’s role in mitochondrial dynamics is well recognized, the molecular mechanism by which it recruits TRAK, and the regulation of this interaction remain incompletely understood. This dissertation investigates the structural and biochemical basis of the MIRO1-TRAK1 interaction and evaluates the roles of MIRO1’s Ca2+- and nucleotide-binding properties in this context. We use cryo-electron microscopy (cryo-EM), site-directed mutagenesis, binding assays, and cellular localization experiments to characterize two distinct binding sites between TRAK1 and MIRO1. The first interaction site (Site-1) involves TRAK1425–428 and maps to a pocket between MIRO1’s second EF-hand pair and C-terminal GTPase. Biochemical reconstitution demonstrates that Site-1 functions independently of TRAK1 dimerization. The second site (Site-2) involves TRAK1569–623 and binds within a cleft formed by MIRO1’s N-terminal GTPase and first EF-hand pair. Site-2 is further stabilized by dimerization through MIRO1’s C-terminal domains and an anti-parallel β-sheet formed by TRAK1 peptides. Both interactions were validated by site-directed mutagenesis and binding assays including affinity pulldowns and isothermal titration calorimetry (ITC). ITC measurements also revealed that two of MIRO1’s EF-hands bind calcium with distinct affinities (3.9 μM and 300 nM), suggesting one may be constitutively bound while the other has potential to be regulated under physiological conditions. High-performance liquid chromatography confirmed both GTPase domains of MIRO1 can exchange GTP for GDP under varying conditions. Despite these capabilities, both TRAK1 binding sites interact with MIRO1 independently of its calcium- or nucleotide-state. Mitochondrial recruitment assays in vitro further demonstrate that both Site-1 and Site-2 are required for proper recruitment of TRAK1 to the outer mitochondrial membrane. This thesis provides the first high-resolution structure of full-length cytosolic MIRO1 in complex with TRAK1, reveals two distinct and cofactor-independent MIRO1-TRAK1 binding sites, and refines current models of mitochondrial motor recruitment. These results establish a foundation for future investigations into the regulation of mitochondrial dynamics and its implications in health and disease.