BACKGROUND: Chaihuang Yishen Granules (CHYS) has been clinically proven to be effective for the treatment of chronic kidney disease (CKD), yet its underlying molecular mechanisms remain largely unexplored. OBJECTIVE: To explore the innovative mechanisms by which CHYS alleviates CKD, focusing on its role in modulating PRDX5/TFAM-mediated mitochondrial homeostasis in renal cells. METHODS: In this study, CKD mouse model was established by unilateral ureteral obstruction (UUO) and adenine (Ade) diet. Treatment interventions were administered by gavage with CHYS at doses of 3.8g/kg (low dose) and 7.6g/kg (high dose). The ameliorative effects of CHYS on CKD were evaluated by changes in renal function, kidney tissue structure, renal fibrosis, and mitochondrial dysfunction markers. Tert‑butyl hydroperoxide (t-BHP)-induced oxidative stress in TCMK1 cells was used to simulate CKD renal fibrosis induced by mitochondrial dysfunction in vitro. RESULTS: CHYS significantly improves renal function and mitigates fibrosis while restoring mitochondrial homeostasis. Notably, PRDX5 expression, which is markedly reduced in CKD patients and mouse models, is substantially upregulated following CHYS treatment. Meanwhile, we demonstrate that ultrasound microbubble-mediated in situ overexpression of PRDX5 confers considerable renal protection in the UUO model. In vitro data show that CHYS effectively prevents t-BHP-induced mtDNA leakage in renal tubular cells, preserving mitochondrial function and stability, an effect compromised by PRDX5 knockdown. Moreover, our protein binding assays uncover a previously unreported interaction between PRDX5 and TFAM, with TFAM knockdown reversing the mitochondrial functional and fibrotic improvements achieved through PRDX5 overexpression and CHYS intervention. CONCLUSION: These findings introduce a pioneering perspective on CHYS's mechanism of action. CHYS enhance TFAM activation through PRDX5 upregulation, counteract ROS-induced mitochondrial damage, and restoring mitochondrial homeostasis, and alleviates the progression of renal fibrosis in CKD, highlighting the innovative therapeutic potential of CHYS in mitochondrial-related renal pathologies.