Objective Optimization of the surgical details of acute myocardial infarction in neonatal 1-day-old mammary mice, and establishment standardized assessment of a stable model. Methods Postnatal day 1 (P1) CD1 mice were subjected to three sets of surgical manipulations. Coronary artery left anterior descending (LAD) ligation (1 mm below the left auricular border, 1 mm in width, and <0. 5 mm ligation depth) was performed in the standard myocardial infarction (SMI) group. The LAD ligation position and width remained unchanged with a >0. 5 mm ligation depth in the deep ligation group (Deep MI, DMI). Only the chest was opened without LAD ligation in the sham surgery group (Sham). The success of LAD ligation was verified by TTC and Evans blue-TTC staining. The extent of myocardial tissue damage, fibrosis, and regeneration of LAD ligation was assessed by HE and Masson staining at 3, 7, 14, 21 and 28 days after surgery. Structural and functional changes of mouse hearts were assessed by echocardiography at 28 days after surgery. Results It describes the process of establishing a P1 neonatal mouse myocardial infarction model in detail and provides stable regeneration and a high survival rate of MI through realization of LAD exposure, ligation, postoperative care, and other processes. Successful ligation of the SMI model was determined by TTC and Evans blue-TTC staining at 1 day postoperatively. Echocardiography at 28 days postoperatively revealed no statistical difference in LVEF, LVFS, LVIDd, or LVIDs compared with the Sham group, indicating that the heart structure and function were restored to normal. Masson staining at 3, 7, 14, 21 and 28 days postoperatively revealed that tissue fibrosis had occurred in 15. 67%, 3. 34%, 2. 99%, 2. 73% and 1. 11% of the heart area, respectively, in the SMI group, indicating almost complete recovery of the myocardial infarct area at 28 days postoperatively. The survival rate of the DMI group was reduced by 35. 71% compared with the SMI group (85. 71% for SMI and 50% for DMI). Ultrasound at 28 days postoperatively showed reduction in LVEF (17. 25±6. 03)%, LVFS (11. 37±4. 06)%, increases in LVIDd (0. 46±0. 15)%, and LVIDs (0. 69±0. 20)% (P<0. 05). The fibrotic area was six times larger in the DMI group than in the SMI group, indicating that complete regenerative repair at 28 days postoperatively could not be achieved with a ligation depth of > 0. 5 mm. Conclusions This study describes in detail the process of establishing a standard acute myocardial infarction model in 1-day-old mice, and evaluated the infarct size and cardiac structure, function, and fibrosis at various time points after surgery by TTC, Evans blue-TTC staining, cardiac ultrasound, and Masson staining. It was clear that complete cardiac repair at 28 days after surgery cannot be achieved with a ligation depth of >0. 5 mm. These data provide a reference to establish a stable and reliable model of acute myocardial infarction in 1-day-old mice for surgical evaluation.