〔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 expression of mitophagy-related markers (Parkin, Pink1, P62, and LC3II) was evaluated via Western blot analysis. 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 restored by NGR1 treatment (P<0.01). The mRNA levels of Parkin, PINK1, and P62 in the H/R group were upregulated compared to the control group (P<0.05), while NGR1 intervention downregulated their expression (P<0.05). Protein expression levels of Parkin, Pink1, and LC3II 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 LC3II while increasing P62 expression (P<0.05). TEM revealed that the mitochondrial structure in the H/R group was severely disrupted, with fragmented and disorganized 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.