Abstract: AIM To investigate the role of nicitinamide riboside (NR) in mitochondrial autophagy and mitochondrial biogenesis in adult mouse cardiomyocytes subjected to high glucose injury. METHODS Adult murine cardiomyocytes were randomly divided into six groups with or without 50 μmol/L nicotinamide riboside for 48 hours. Cell apoptosis was detected by TUNEL and flow cytometry and mitochondrial membrane potential was assessed by JC-1. The protein expressions of peroxisome proliferator-activated receptor γ co-activator (PGC)-1α, Tfam, LC3, Atg5, p62, p53 and Parkin were analyzed by Western blot. RESULTS Compared with those in the control group, high glucose significantly increased the cell apoptosis index (P<0.01) and reduced the mitochondrial membrane potential (P<0.01), as well as the expressions of PGC-1α, Tfam, LC3, Atg5, p53 and Parkin (P<0.01). However, the expression of P62 significantly increased (P<0.01). Additionally, NR decreased the cell apoptosis index (P<0.01) and increased the mitochondrial membrane potential (P<0.01). The mitochondrial membrane potential was reversed after knockdown of PGC-1α by siRNA, knockdown of Sirt3 by siRNA, or adding nutlin-3, a p53 agonist. NR also increased the expression of PGC-1α (P<0.01), which was reversed after knockdown of PGC-1α by siRNA. NR increased the expressions of LC3, Atg5, p53 and Parkin (P<0.01). These proteins were reversed after knockdown of Sirt3 by siRNA or adding nutlin-3, while the expression of p62 decreased (P<0.01). CONCLUSION Nicotinamide riboside, as a potential treatment, could improve mitochondrial biogenesis via up-regulating the expression of PGC-1α and improve mitochondrial autophagy via up-regulating the expression of p53, thus potentially reducing high glucose injury in adult murine cardiomyocytes.