Objective: To investigate the role and mechanism of the tRNA-derived fragment AsnGTT-33 (tRF-AsnGTT-33) in podocyte injury within a murine model of adriamycin(ADR)-induced nephropathy. Methods: An ADR nephropathy mouse model was established via a single tail vein injection of ADR(10 mg/kg). tRF-AsnGTT-33 adeno-associated virus(AAV)(0.5 mL per mouse)was administered via tail vein to achieve in vivo overexpression. Renal function was assessed by measuring serum creatinine (Scr) levels using the sarcosine oxidase method and blood urea nitrogen (BUN) levels using the urease method. Renal pathological injury was evaluated by hematoxylin and eosin staining(HE) and periodic acid-schiff staining(PAS) to observe glomerular and tubular damage. The mRNA and protein expression levels of the podocyte markers nephrin and podocin were analyzed by quantitative PCR(qPCR)and Western blot, respectively. In vitro, podocyte injury was induced by 1 mg/L ADR. Following transfection with a tRF-AsnGTT-33 mimic, the expression levels of nephrin and podocin were detected by qPCR and Western blot. The miRDB database was used to predict Ras-related protein RAB21 as a potential target gene. The effect of tRF-AsnGTT-33 overexpression on RAB21 expression was subsequently examined in both cultured podocytes and mouse renal tissues. Results: Renal tissues from ADR nephropathy mice exhibited significantly downregulated tRF-AsnGTT-33 expression,accompanied by markedly elevated Scr and BUN levels, reduced expression of nephrin and podocin, and aggravated renal pathological injury, including thickening of the glomerular basement membrane, intratubular protein casts, and interstitial fibrosis. Overexpression of tRF-AsnGTT-33 significantly lowered Scr and BUN levels, alleviated pathological damage, and upregulated the expression of nephrin and podocin. In ADR-induced podocytes, tRF-AsnGTT-33 expression was decreased, along with reduced nephrin and podocin expression; these effects were reversed by tRF-AsnGTT-33 overexpression. Furthermore, tRF-AsnGTT-33 overexpression led to a significant downregulation of RAB21 expression in both podocytes and mouse renal tissues. Conclusion: tRF-AsnGTT-33 may alleviate ADR-induced podocyte injury by targeting RAB21, providing a theoretical foundation for identifying novel therapeutic targets for chronic kidney disease.