Notoginsenoside R1 (NGR1) is separate from Panax notoginsenosides (PNS), and plays a role similar to phytoestrogen in preventing and treating cardiovascular diseases. However, the protective mechanism of NGR1 in the myocardial ischemia/reperfusion injury via the estrogen receptor (ER) pathway remains unclear, which hinder its application. This study aimed to study the preventive mechanisms of NGR1 in the apoptosis of H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R). NGR1 did not affect the expression of ERα and ERβ proteins in normal H9c2 cardiomyocytes. However, NGR1 could upregulate the ERα and G protein-coupled receptor 30 (GPR30) proteins in H9c2 cardiomyocytes after H/R without affecting ERβ levels. Moreover, it significantly affected the expression levels of PI3K and its downstream apoptosis proteins such as Bcl-2 Associated X Protein (Bax), B cell lymphoma/lewkmia-2 (Bcl-2), caspase-3, and so forth. Whereas, after adding the PI3K protein antagonist, the modulatory expression levels of PI3K and its downstream apoptosis proteins were remarkably abolished. After adding ERα and GPR30 antagonists, NGR1 had no significant effect on the expression of PI3K and its downstream Akt protein in the model group. The data of flow cytometry showed that after adding the ERα, GPR30 and PI3K antagonists, the apoptotic rate of cardiomyocytes had no significant changes compared with the model group. This study demonstrated that NGR1 protected H9c2 cardiomyocytes from the injury after H/R by affecting ERα and GPR30 to regulate the expression levels of PI3K and its downstream apoptosis proteins.