Aim To investigate the mechanism of microRNA-301 (miR-301) influencing cardiomyocyte apoptosis in rats with coronary heart disease (CHD). Methods SD rats were randomly divided into control group, model group, miR-301 agonist group (agomir miR-301 group), miR-301 agonist control group (agomir NC group), Wnt /β-catenin pathway inhibitor group (Dkk1 group), and agomir miR-301＋Dkk1 group, 10 rats in each group. Except for the control group, the other groups were fed with high-fat diet to establish a CHD model, and after 1 week of continuous administration, ultrasound was used to detect the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (FS), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD) and other cardiac function indicators in rats. HE staining was used to observe the morphology of rat myocardium, TUNEL was used to detect cardiomyocyte apoptosis, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression of miR-301 in myocardial tissue, Western blot was used to detect the protein expression of Caspase-3, Bax, Wnt3a and β-catenin in rat myocardial tissue. Results Compared with the control group, LVEF and FS in model group decreased by 49.2% and 49.1%, the expression level of miR-301 in myocardial tissue decreased by 69.0%, and the protein expression levels of Wnt3a and β-catenin decreased by 60.7% and 39.7% (P＜0.05). LVEDD and LVESD increased by 32.2% and 99.6%, the apoptosis rate of cardiomyocytes increased by 1362.6%, and the expression levels of Caspase-3 and Bax proteins in myocardial tissue increased by 100.0% and 114.6% (P＜0.05). Compared with agomir miR-301 group, LVEF and FS in agomir miR-301 group increased by 71.4% and 71.8%, the expression level of miR-301 in myocardial tissue increased by 1935.5%, and the protein expression levels of Wnt3a and β-catenin increased by 102.4% and 45.1% (P＜0.05), LVEDD and LVESD decreased by 15.6% and 39.2%, the apoptosis rate of cardiomyocytes decreased by 65.0%, and the expression levels of Caspase-3 and Bax in myocardial tissue decreased by 70.2% and 78.4% (P＜0.05), while Dkk1 could reverse this phenomenon. Conclusion MiR-301 can improve cardiomyocyte apoptosis in rats with coronary heart disease by activating Wnt/β-catenin signaling pathway.