Eukaryotic cells utilize membrane contact sites (MCSs) to mediate communication between organelles, with mitochondria-ER contact sites (MERCs) being among the most extensively studied due to their essential roles in maintaining cellular homeostasis. MERCs facilitate critical processes, including lipid biosynthesis, calcium transfer, and mitochondrial dynamics, via specific protein complexes. Dysregulation of MERCs has been implicated in the pathogenesis of several neurodegenerative diseases. Mitofusin 2 (MFN2), proposed to reside on the ER, is hypothesized to form heterotypic dimers with mitochondrial MFN1/2, enabling the transport of calcium and phosphatidylserine. However, its ER localization remains controversial, as super-resolution imaging has yet to provide definitive evidence. This uncertainty raises a key question: Are other ER-resident proteins involved in regulating the formation and function of MERCs? To resolve this ambiguity, it is necessary to identify novel MFN2-interacting proteins on the ER and examine their roles in MERC formation and function. Our study identified REEP5, a member of the ER membrane-shaping protein family, as a key mediator of ER-mitochondria tethering through a targeted screen and demonstrated its specific interaction with mitochondria-localized MFN1/2 at MERCs. The interaction involves the C-terminal cytoplasmic domains of REEP5 and MFN1/2. Further analysis revealed that REEP5-MFN1/2 facilitates the coordinated movement of mitochondria with the tubular ER and plays a critical role in regulating the intracellular distribution of mitochondria. Results demonstrated that attenuated interactions between REEP5 and MFN1/2 lead to the collapse of mitochondrial networks and perinucleus clustering. Conversely, enhanced interactions between REEP5 and MFN1/2 lead to sustained mitochondrial attachment to the tubular ER, thereby inducing mitochondrial hyperfusion. These findings underscore the importance of balanced REEP5-MFN1/2 interactions for proper cytoplasmic distribution of mitochondria. Furthermore, this study further elucidates the role of the REEP5-MFN1/2 complex in regulating mitochondrial reactive oxygen species (ROS) homeostasis. Previous reports have linked mitochondrial distribution patterns with the changes in ROS levels, and REEP5 reduction is found to induce cytosolic ROS accumulation. We found that the loss of either REEP5 or MFN1/2 resulted in increased mitochondrial ROS levels, whereas enhancing the interaction between REEP5 and MFN1/2 significantly reduced mitochondrial ROS levels. These results indicated that the interaction of REEP5-MFN1/2 is critical for mitochondrial ROS homeostasis. Overall, this study is the first to identify a novel complex consisting of REEP5 and MFN1/2, which plays a critical role in regulating mitochondrial distribution and ROS homeostasis.