The intestinal epithelium relies on autophagy to maintain barrier integrity and homeostasis, and dysregulation of this process has been implicated in inflammatory bowel disease (IBD). While tRNA-derived small RNAs (tsRNAs) are emerging as regulators of cellular stress responses, their roles in intestinal autophagy remain poorly understood. Here, we identify that 5'-tiRNA-Lys, a tsRNA derived from mature tRNA-Lys, is significantly upregulated in the inflamed intestinal epithelium of IBD patients. Overexpression of 5'-tiRNA-Lys in mice ameliorates dextran sulfate sodium (DSS)-induced colitis. Mechanistically, 5'-tiRNA-Lys enhances autophagy in intestinal epithelial cells (IECs), as evidenced by elevated LC3-II level and autophagosome formation. RNA pull-down and immunoprecipitation assays reveal that 5'-tiRNA-Lys directly binds to the RNA-binding protein EWSR1 via its RNA recognition motif, disrupting EWSR1's interaction with the Drosha/DGCR8 microprocessor complex. This interference specifically suppresses the maturation of miR-125a, a microRNA that targets the autophagy-promoting gene UVRAG. Consequently, 5'-tiRNA-Lys increases UVRAG expression, thereby enhancing autophagic activity. Our findings reveal that 5'-tiRNA-Lys modulates autophagy through EWSR1-mediated miR-125a processing, which in turn affects intestinal inflammation, highlighting the potential of 5'-tiRNA-Lys as a therapeutic target for IBD.