To investigate the effect of hydroxycamptothecin liposomes (HCPTL) on renal mitochondrial dynamics in obstructive nephropathy rats based on the miR-20b-5p/thioredoxin interacting protein (TXNIP) pathway. After successfully constructing a rat model of obstructive nephropathy by unilateral ureteral ligation (UUO) method, the rat model was intervened with hydroxycamptothecin (HCPT), HCPTL, and HCPTL+miR-20b-5p antagomir (miR-Ant) , respectively, and the serum creatinine (SCR) and blood urea nitrogen (BUN) levels were detected. The changes of renal fibrosis and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were observed. Mitochondrial structure, mitochondrial membrane potential, mitochondrial fusion protein 2 (MFN2), dynamic related protein 1 (DRP1) average optical density, expression of miR-20b-5p and changes of TXNIP, MFN2 and DRP1 proteins were evaluated in renal tissue. The results showed that after HCPT and HCPTL intervention, UUO rats showed a reduction in blue stained fibrotic areas, the swelling of mitochondrial matrix and rupture of cristae were alleviated, the SCR and BUN in serum decreased, the SOD level, mitochondrial membrane potential, average optical density of MFN2, expression of miR-20b-5p, and expression of MFN2 protein in renal tissue increased, the MDA level, average optical density of DRP1, expression of TXNIP, and DRP1 proteins in renal tissue decreased, and the corresponding change trend was more obvious after HCPTL intervention (P<0.05); miR-Ant weakened the improvement effect of HCPTL on renal mitochondrial dynamics in obstructive nephropathy rats. The above results show that HCPTL may improve renal mitochondrial dynamics, enhance antioxidant stress capacity, and inhibit renal injury in obstructive nephropathy rats by regulating the miR-20b-5p/TXNIP pathway.