Cardiosphere-derived cells (CDCs) are cardiac progenitor/stromal cells with immunomodulatory, anti-fibrotic, and pro-regenerative properties. These therapeutic actions antagonize crucial pathways central to the pathology of Duchenne muscular dystrophy (DMD). Indeed, the HOPE-2 trial showed human allogeneic CDCs to be safe and to preserve cardiac and upper limb function in advanced-stage DMD patients. Mechanistic studies in preclinical models demonstrate CDCs work indirectly by secreting extracellular vesicles (CDC-EVs), which are lipid nanoparticles laden with a rich repertoire of bioactive molecules. In CDC-EVs, the largest percentage of mapped reads are transfer RNA (tRNA) fragments and halves. These molecular entities were previously thought to be nonspecific degradation products, but they are increasingly recognized as comprising a novel class of small ncRNAs with potential therapeutic bioactivity. In CDC-EVs, a species comprising the 5’ half of one specific tRNA is particularly plentiful, we call tREX-1. When packaged into a transfection reagent and delivered intravenously to mdx mice, tREX-1 has potent disease-modifying bioactivity: key disease manifestations of DMD, including structural and functional abnormalities in heart and skeletal muscle, are partially but significantly reversed. However, this effect is abrogated when macrophages are depleted prior to tREX-1 infusion, suggesting macrophages are primary mechanistic targets of tREX-1. In macrophages, tREX-1 induces major transcriptomic changes both in vitro and in vivo, leading to functional reprogramming. Thus, tREX-1 serves as the prototype for a new class of small ncRNA drugs, which may be complementary to (and potentially synergistic with) exon-skipping agents and emerging gene therapies.