〔Abstract〕 To investigate the mechanism by which Notoginsenoside R1（NGR1）ameliorates hypoxia/reoxygenation（H/R）-induced injury in AC16 human cardiomyocyte cell lines through the regulation of mitophagy. Methods Common genes linked to hypoxia/reoxygenation injury and mitophagy were identified by intersecting data from GeneCards and MitoCarta databases. AC16 cell viability was assessed via CCK-8 assay under varying NGR1 concentrations（0，6. 25，12. 5，25，50，100，200，300，400，500 μmol/L）. AC16 cells were divided into the following groups：control group（Control），model group（H/R），and treatment groups（H/R + NGR1 at 100， 200，and 300 μmol/L）. Mitochondrial membrane potential ( ΔΨm）was measured using 5，5'，6，6'-tetrachloro-1， 1'，3，3'-tetraethylbenzimidazolylcarbocyanine iodide（JC-1）staining. Transcriptional levels of mitophagy-related genes（Parkin，Pink1，P62）were quantified by reverse transcription-quantitative PCR（RT-qPCR）. Protein ex- pression of mitophagy-related markers（Parkin，Pink1，P62，and LC3BII）was evaluated via Western blot analy- sis. Mitochondrial ultrastructure was visualized by transmission electron microscopy（TEM）. Results Compared to the control group，cell viability in the H/R group significantly decreased（P<0. 01）. Treatment with NGR1 at concentrations above 100 μmol/L significantly enhanced the cell viability of AC16 cells compared to the H/R group （P<0. 01）. H/R induced a significant decrease in mitochondrial membrane potential（P<0. 01）， which was re- stored by NGR1 treatment（P<0. 01）. The mRNA levels of Parkin，Pink1，and P62 in the H/R group were upregu- lated compared to the control group（ P<0. 05）， while NGR1 intervention downregulated their expression（ P< 0. 05）. Protein expression levels of Parkin，Pink1，and LC3BII in the H/R group significantly increased，while P62 expression decreased compared to the control group（P<0. 01）. In contrast，different doses of NGR1 treatment significantly reduced the expression of Parkin，Pink1，and LC3BII while increasing P62 expression（P<0. 05）. TEM revealed that the mitochondrial structure in the H/R group was severely disrupted，with fragmented and disor- ganized cristae，which was alleviated by NGR1. Conclusion NGR1 ameliorates H/R-induced AC16 cell injury， and its mechanism may be associated with modulating the Pink1/Parkin pathway to suppress excessive mitophagy.