Background: Acute myocardial infarction (AMI) induces significant myocardial damage, ultimately leading to heart failure as the surrounding healthy myocardial tissue undergoes progressive deterioration due to excessive mechanical stress. Methods: This study aimed to investigate myocardial regeneration in a porcine model of AMI using an acellular amniotic membrane with fibrin—termed an amnion bilayer (AB) or heart patch—as a cellular delivery system using porcine amniotic stem cells (pASCs) and autologous porcine cardiomyocytes (pCardios). Fifteen pigs (aged 2–4 months, weighing 50–60 kg) were randomly assigned to three experimental groups (n = 5): control group (AMI induction only), pASC group (pASC transplantation only), and coculture group (pASC and pCardio transplantation). AMI was induced via posterior left ventricular artery ligation and confirmed through standard biomarkers. After eight weeks, histological and molecular analyses were conducted to assess myocardial regeneration. Results: Improvement in regional wall motion abnormality (RWMA) was observed in 60% of the coculture group, 25% of the pASC group, and none in the control group. Histological analysis of the control group revealed extensive fibrosis with pronounced lipomatosis, particularly at the infarct center. In contrast, pASC and coculture groups exhibited minimal fibrotic scarring at both the infarct center and border regions. Immunofluorescence analysis demonstrated positive α-actinin expression in both the pASC and coculture groups, with the coculture group displaying sarcomeric structures—an organization absent in control group. RNA expression levels of key cardiomyogenic markers, including cardiac troponin T (cTnT), myosin heavy chain (MHC), and Nkx2.5, were significantly elevated in the treatment groups compared to the controls, with the coculture group exhibiting the highest MHC expression. The expression of c-Kit was also increased in both treatment groups relative to the control. Conversely, apoptotic markers p21 and Caspase-9 were highest in the control group, while coculture group exhibited the lowest p53 expression. Conclusion: Epicardial transplantation of an acellular amniotic heart patch cocultured with cardiomyocytes and pASCs demonstrated superior cardiomyogenesis after eight weeks compared to pASC transplantation alone or control conditions. The coculture system was found to enhance the cardiac regeneration process, as evidenced by improved RWMA, distinct sarcomeric organization, reduced fibrotic scarring, and lower apoptotic gene expression. © The Author(s) 2025.