Objective: To investigate the effect of mitochondrial targeting drug Mitochonic acid 5 (MA-5) on renal fibrosis and its mechanism. Methods: Twenty-four 8-week-old SPF C57BL/6J male mice were randomly divided into four groups: control group, MA-5 group, unilateral ureteral ligation (UUO) group and UUO+MA-5 group. The ureter and kidney were exposed by modeling operation, and the UUO group and the UUO+MA-5 group were sutured after ligation,while the other two groups were directly sutured after exposure. From the 2nd day after operation, the MA-5 and the UUO+MA-5 group were given MA-5 by gavage continuously until the 7th day, and the control and UUO group were given corn oil by gavage. The mice were sacrificed on the 7th day after UUO, and the kidney and blood samples were collected. Subsequently, Masson and Sirius Red staining were used to detect the degree of renal fibrosis. The expression of α-SMA and Collagen I were explored by immunohistochemistry. Furthermore, Western Blot was used to detect the expression of mitochondrial related proteins. Mouse renal tubular epithelial cells were cultured in vitro and stimulated with transforming growth factor-β (TGF-β). Western Blot was used to detect the expression of α-SMA, Fibronectin and Vimentin in tubular epithelial cells with or without MA-5 intervention. Results: Seven days after UUO, Masson and Sirius Red staining of renal tissue showed that the UUO group had severe renal fibrosis, and the UUO+MA-5 group had significantly reduced renal fibrosis compared with the UUO group. The results of Western Blot and immunohistochemistry showed that the expression of α-SMA and Collagen I in the UUO+MA-5 group was significantly lower than that in the UUO group (p<0.05). Further study showed that mitochondrial biosynthesis, fusion and movement were decreased in the UUO group, and the expression of manganese superoxide dismutase (SOD2) was decreased. MA-5 treatment significantly increased the expression of Mitofilin in the kidney of UUO mice, improved mitochondrial function, and increased the expression of PGC1-α, Mfn1, Miro1 and SOD2 (p<0.05). The results in vitro showed that MA-5 could reduce the expression of fibrosis-related proteins induced by TGF-β in cultured tubular epithelial cells (p<0.05). Conclusions: Renal fibrosis occurs in mice after UUO, and MA-5 can reduce renal fibrosis by maintaining mitochondrial homeostasis. In vitro experiments showed that MA-5 could attenuate TGF-β induced tubular epithelial cells fibrosis.