Abstract: AIM To determine the role of Parkin-mediated mitophagy in cardiomyocytes exposed to high glucose and saturated fatty acid stimulation. METHODS Neonatal mouse ventricular myocytes were separated and cultured in DMEM with normal (5.5 mmol/L) dose of glucose. LV-LacZ or LV-Parkin was transfected into cardiomyocytes. After transfected for 48 hrs, cardiomyocytes were cultured in DMEM with normal (5.5 mmol/L) or high dose of glucose (25 mmol/L HG) and palmitate (16:0; 500 μmol/L HF) for 24hrs. So we divided the experiment into four groups: ①NG-LacZ; ②NG-Parkin; ③HG-HF-LacZ; ④HG-HF-Parkin. The expression of proteins was analyzed by Western blot and the number of autophagosomes was counted by immunofluorescence. The mitochondrial membrane potential was measured by JC-1 staining and the apoptotic index was evaluated by TUNEL. RESULTS Compared with that in control group, mitophagy associated protein Parkin was significantly reduced by high glucose and high palmitate( P<0.05), suggesting that mitophagy might be reduced in this condition. The expression of LC3Ⅱ was significantly increased (P<0.05), indicating the accumulation of autophagosomes. Up-regulated P62 (P<0.05) suggested that HG and HF resulted in impaired clearance of autophagosomes. Compared with those in HG-HF group,,LV-Parkin up-regulated the expression of Parkin and LC3Ⅱ (P<0.05) and decreased the level of P62 significantly (P<0.05) following HG-HF treatment, indicating that Parkin over-expression enhanced mitophagy and autophagy flux. Parkin reduced the apoptosis (P<0.05) caused by high glucose and high plamitate in cardiomyocytes. CONCLUSION Parkin-mediated mitophagy plays a protective role in cardiomyocyte injury induced by high glucose and high palmitate.