Activation of inflammation is an important pathogenic factor contributing to the development of chronic kidney disease (CKD). Recent studies manifested the implication of impaired mitophagy mediated NLRP3 inflammasome activation in the progression of CKD. Mitochondria-targeted antioxidant mitoTEMPO showed antioxidant and anti-inflammatory properties in kidney disease. This study aims to investigate the protective mechanism of mitoTEMPO on podocyte injury related to mitophagy and NLRP3 inflammasome. Our results showed that mitoTEMPO obviously ameliorated renal function and podocyte injury in CKD model rats induced by cationic bovine serum albumin (C-BSA). More importantly, mitoTMEPO significantly inhibited NLRP3 inflammasome activation compared with CKD model rats (P < 0.01). In vitro, TNF-alpha damaged human podocyte cells (HPC) and activated NLRP3 inflammasome, which was rescued by NLRP3 inhibitor and mitoTEMPO. Meanwhile, mito-TEMPO lessened excessive mitochondrial ROS (mtROS) and degressive mitochondrial membrane potential (MMP) in HPC. We also found that mitoTEMPO induced mitophagy in vivo and in vitro. Moreover, silenced Parkin dramatically reserved the inhibitory effect of mitoTEMPO on NLRP3 inflammasome. Taking together, these findings reveal that mitoTEMPO ameliorated podocyte injury by inhibiting NLRP3 inflammasome via PINK1/ Parkin pathway-mediated mitophagy. MitoTEMPO may be a new candidate to protect against podocyte injury in CKD.