Ginsenoside Rb3 (GRb3) is an active ingredient extracted from Panax ginseng, which is known to enhance blood supply to heart and treat a variety of cardiac diseases. The aim of this study was to investigate the effect of GRb3 on myocardial injury induced by sleep deprivation and its mechanisms. GRb3 20 mg/kg group showed lower heart rate ((438.20 ± 10.06) bpm), creatine kinase ((27.43 ± 2.85) U/mg pro) and lactate dehydrogenase ((412.90 ± 35.65) U/L) than SD group ((592.00 ± 7.78) bpm, (44.18 ± 1.24) U/mg pro, (526.60 ± 38.18) U/L). GRb3 improved myocardial tissue damage and abnormal mitochondrial morphological changes. GRb3 also reduced the abnormal proteins expression of atrial natriuretic peptide (ANP), sirtuin 3 (SIRT3), tumor protein P53 (P53), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), transferrin receptor (TFRC), acyl-CoA synthetase long chain family member 4 (ACSL4), heme oxygenase-1 (HO-1), ferritin heavy chain 1 (FTH1), ferroptosis suppressor protein 1 (FSP1). GRb3 ameliorated oxidative stress, such as malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD). GRb3 also decreased reactive oxygen species (ROS) content, restored cellular abnormal mitochondrial morphology and reversed the expression of ferroptosis-related proteins on H9c2 cells pretreated with Erastin. While the above effect could be reversed by 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP). In summary, GRb3 exerts cardioprotective effects by regulating SIRT3/P53/SLC7A11/GPX4 axis to inhibit myocardial ferroptosis in sleep deprived mice. The findings imply that GRb3 holds promising potential for innovative approaches to safeguarding human cardiac health.