Abstract Body (Do not enter title and authors here): Heart failure (HF) is an increasingly prevalent cause of morbidity and mortality in the United States, with a projected prevalence of 8.7 million by 2030. Cardiac fibrosis, which develops following most myocardial injuries, significantly contributes to HF progression. Despite this critical clinical importance, there are currently no FDA-approved anti-fibrotic drugs to prevent HF development. Transfer RNA-derived small RNAs (tsRNAs, or tDRs), generated through tRNA cleavages, represent an emerging class of regulatory molecules that control adaptive stress response. Our previous study established a comprehensive stress-specific tDR atlas and identified hundreds of ischemia-regulated tDR in cardiac fibroblasts (CFs). However, their functions remain to be explored. Here, we found that the biogenesis of Asp-GTC-3’tDR, derived from the 3′ end of tRNA-Asp-GTC, is promptly enhanced by ischemia in both cultured CFs and mouse cardiac ischemic injury models, but suppressed in myofibroblasts and in failing heart tissues. Introducing Asp-GTC-3’tDR to cultured CFs markedly downregulated fibrotic gene expression and attenuated TGFβ-induced CF activation. Delivery of Asp-GTC-3’tDR mimics to cardiac tissues using polymer nanoparticles significantly mitigated cardiac fibrosis, alleviated adverse cardiac remodeling, and improved cardiac functions in the mouse myocardial infarction model. Conversely, silencing cardiac Asp-GTC-3’tDR using an optimal antisense oligonucleotide promotes fibrotic responses in healthy heart tissues, confirming its protective role. Mechanistically, transcriptomic analysis indicated that Asp-GTC-3’tDR suppresses TGFβ signaling and extracellular matrix assembly pathways while activating autophagy. Functional studies confirmed that Asp-GTC-3’tDR overexpression activates autophagic flux, while its inhibition blocks autophagic flux. Proteomic analysis of Asp-GTC-3’tDR-binding partners identified ribosomal proteins as primary interacting proteins. Further characterization indicated that Asp-GTC-3’tDR interacts with stalled ribosomes and promotes the formation of stress granules, which are eventually targeted to the autophagy pathway for degradation. Together, we identified an ischemia-induced tDR, Asp-GTC-3’tDR, that significantly attenuates cardiac fibrosis both in cellular and murine models by enhancing autophagic flux. These findings establish Asp-GTC-3’tDR as a promising novel therapeutic target for treating cardiac fibrosis and HF progression.