<p>Mitochondrial dysfunction underlies the major defect in muscle atrophy (characterized by the loss of skeletal muscle mass and function). Mesenchymal stem cells (MSCs), which can mediate mitochondrial transfer (MT) via tunneling nanotubes (TNTs), have been shown to exert therapeutic effects, yet the underlying mechanism remains unclear. Mitochondrial Rho GTPase 1 (Miro1) is crucial for regulating mitochondrial homeostasis; in this study, we aimed to investigate the roles of Miro1 and Milton in MSC-based therapy for muscle atrophy. Dexamethasone (DEX)-induced C2C12 cells and chronically aged mice were used as in vitro cellular and in vivo muscle atrophy models, respectively. In vitro experiments demonstrated that overexpression of Milton alone failed to enhance MT in DEX-induced C2C12 cells. Although Milton could promote the formation of TNTs, it was unable to drive mitochondrial movement along microtubules in the absence of Miro1.In contrast, Miro1 knockdown (MSCmiro1^Lo) significantly reduced MT in vivo, while Miro1 overexpression (MSCmiro1^Hi) improved mitochondrial morphology, increased muscle fiber count and cross-sectional area, upregulated the expression of type I/III collagen, and downregulated the expression of Atrogin1 and MURF1. Additionally, Miro1 overexpression ameliorated functional outcomes such as grip strength, running distance, and physical activity, and elevated the levels of proteins related to mitochondrial fusion, mitophagy, and biogenesis in damaged muscle cells. These findings indicate that Miro1 is a critical driver of MT, and Miro1-enhanced MT confers substantial in vivo therapeutic benefits. This study provides robust evidence supporting Miro1 as a potential target for the treatment of muscle atrophy-related disorders.</p>