<p>In cardiovascular research, melatonin has shown promise in exhibiting antifibrotic properties and modulating endoplasmic reticulum (ER) stress. However, the exact mechanism by which it influences myocardial fibrosis has not been fully clarified. Therefore, this research aimed to investigate the inhibitory effect of melatonin on the progression of myocardial fibrosis through a mechanism involving the BIP/PERK/CHOP signaling pathway, both in silico and in vivo experimental models. In in silico studies, molecular docking and molecular dynamics simulations were employed to predict the binding affinity of melatonin to ER stress arm proteins, BIP, and PERK. Following, in vivo experiments were carried out to confirm in silico analyses. In animal studies, rats were administered melatonin intraperitoneal (10 mg/kg per day) for 3 weeks, and on the 6th and 7th days, they were given isoproterenol at a dose of 170 mg/kg subcutaneous to estabilish myocardial fibrosis model. The morphological changes in cardiac tissue were assessed using hematoxylin and eosin (H&amp;E) and Masson’s trichrome staining. Additionally, the expression of BIP and CHOP, a key downstream target of the PERK pathway, was analyzed through real-time PCR and immunohistochemistry. In silico studies suggest melatonin interacts with BIP and PERK, demonstrating strong binding energy and forming a stable complex with both proteins. However, its affinity and stability with PERK are greater than with BIP. Furthermore, immunohistochemistry and qRT-PCR findings indicated that melatonin notably downregulated the expression of BIP and CHOP in the isoproterenol-induced cardiac fibrosis model. The strong binding affinity of melatonin for BIP and PERK, coupled with its impact on the downregulation of BIP and CHOP proteins in the isoproterenol-induced cardiac fibrosis model, suggests that melatonin’s antifibrotic effects on myocardial tissue may be related to its ER stress inhibitory effects.</p> Graphical Abstract <p></p>