Duchenne muscular dystrophy (DMD) is a myodegenerative disease associated with inflammation, myocardial fibrosis, and systolic dysfunction, which has little therapies proven to benefit the heart. Cardiosphere derived cells (CDCs) are progenitor/stromal cells of cardiac origin with disease-modifying bioactivity in mice and humans with DMD. Mechanistic studies showed CDCs work by secreting extracellular vesicles (EVs), lipid nanoparticles enriched with non-coding RNA (ncRNA). One such ncRNA, we call tREX-1, is a 5’ half of a transfer RNA (tRNA) and is plentiful in CDC-EVs. tREX-1, on its own, is bioactive in macrophages, an immune cell implicated in DMD pathophysiology. In mdx mice, a mouse model of DMD, tREX-1 preserved heart function, which was decreased in control mice after 4 weeks of administration. This effect was associated with a reduced myocardial fibrosis, a key manifestation of DMD in mice and humans. RNA-seq showed tREX-1 to be immunomodulatory in the heart of mdx mice. To better understand tREX-1 cardio-immunology, we conducted single-cell RNA-seq on cells isolated from mdx mice previously administered tREX-1 or scrambled RNA control. Single cell RNA-seq showed tREX-1 boosts the number of reparative CCR2- resident cardiac macrophages and reprograms monocyte-derived macrophages towards a wound healing phenotype. Flow cytometry revealed tREX-1 expands the pool of CCR2-/MHC-IIlow resident cardiac macrophages, a population of embryonic origin shown to be critical for the repair of neonatal mouse hearts following myocardial infarction. We next depleted macrophages using clodronate liposome and administered tREX-1 or scramble. When macrophages are absent, tREX-1 is ineffective. In summary, these data show tREX-1 exerts disease-modifying bioactivity in mice with DMD by modulating the innate immune response and motivate further testing of tREX-1 as a next-generation therapeutic for DMD.