Objective To observe the effects of growth differentiation factor-15 (GDF-15) on collateral circulation and cardiac function in rats with acute myocardial infarction (AMI) in relation to the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathway. Methods An AMI rat model was constructed by ligating the left anterior descending coronary artery. After modeling, the rats were divided randomly into Sham, Model, and GDF-15 groups ( n=12 rats per group). Rats in the GDF-15 group were injected intraperitoneally with recombinant GDF-15 protein, and the other two groups were injected with the same amount of normal saline twice a week for 8 consecutive weeks. Cardiac function was detected by echocardiography. Pathological damage to rat myocardial tissue was detected by hematoxylin and eosin staining and the collateral circulation was observed by CD31 immunohistochemical staining. Vascular endothelial growth factor (VEGF) mRNA expression was detected by quantitative polymerase chain reaction. Transcriptomic sequencing of heart tissues in the model and GDF15 groups was performed and differentially expressed genes (DEGs) were screened. Pathway enrichment analysis of the DEGS was carried out according to the Kyoto Encyclopedia of Genes and Genomes (KEGG). Nitric oxide (NO), reactive oxygen species (ROS), and cGMP were detected using kits, and VEGF, endothelial nitric oxide synthase (eNOS) monomer, p-eNOS^ser1177 monomer, eNOS dimer, and PKG protein were detected by Western blot. Results Left ventricular end-systolic diameter (LVEDs) and left ventricular end-diastolic diameter (LVEDd) were increased (P<0.001), and left ventricular ejection fraction (LVEF) and the short-axis shortening rate (FS) were decreased in the Model group compared with the Sham group(P<0.001). Myocardial cell necrosis was more severe, vascular density in the infarcted area was decreased(P<0.05), but VEGF mRNA and protein levels were no change(P>0.05), and levels of NO, eNOS dimer, cGMP, and PKG protein were decreased(P<0.05), and expression levels of ROS, eNOS monomer, and p-eNOS^ser1177 monomer were increased (P<0.05). LVEDs and LVEDd decreased (P<0.05), LVEF and FS increased(P<0.01), myocardial cell necrosis was relieved, vascular density in the infarcted area increased significantly(P<0.0001), and VEGF mRNA levels increased(P<0.0001), compared with the Model group. Transcriptomic sequencing identified 324 DEGs, including 230 up-regulated and 94 down-regulated genes. According to KEGG enrichment analysis, the cGMP-PKG signaling pathway showed the most significant difference in the T20 pathway. VEGF, NO, eNOS dimer, cGMP, and PKG protein levels were all increased(P<0.05), while ROS, eNOS monomer, and p-eNOS^ser1177 monomer were decreased in the GDF-15 group (P<0.05). Conclusions GDF-15 can promote collateral circulation in ischemic myocardium and improve cardiac function by inhibiting eNOS decoupling and activating the NO/cGMP/PKG pathway.