Atherosclerosis is a common chronic disease characterized by the formation of atheromatous plaques and endothelial dysfunction. Endothelial-to-mesenchymal transition (EndMT) has been identified as a crucial driver of atherosclerosis, with TGF-β serving as a pivotal mediator of EndMT. Isoforskolin (ISOF), derived from the plant Coleus forskohlii, is an effective activator of adenylyl cyclase (AC). AC can catalyze the production of cyclic adenosine monophosphate (cAMP), mediating various biological functions. Several phosphodiesterase (PDE) inhibitors that degrade cAMP have been clinically utilized in the treatment of atherosclerosis. However, the evidence regarding the efficacy and mechanisms of AC agonists in the treatment of atherosclerosis remains inadequate.In this study, our primary objective was to examine the therapeutic impact of ISOF on atherosclerosis and elucidate its potential mechanisms.Male ApoE-/- rats were fed a high-fat diet for 18 weeks and then administered ISOF by gavage continuously for 12 weeks. A cell model was established by injuring mouse aortic endothelial cells (MAECs) with ox-LDL. Oil Red O staining and Masson staining were used to assess the plaque area and content of collagen. Aortic vasodilatory function was analyzed using the DMT Myograph system. Immunofluorescence was used to determine the localization of CD31 and α-SMA. Proteomics analysis was utilized to identify potential pharmacological mechanisms of ISOF. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to detect the mRNA expression levels of AC (1-10) in aortic tissue. AC activity and cAMP concentration were detected using specific kits. The CCK-8 assay, wound-healing, and transwell assays were used to measure cell viability and proliferation. Mechanistically, western blot analysis was used to detect candidate protein expression levels. Finally, the pharmacological knockdown of AC5 was employed to clarify the potential mechanism of ISOF.ISOF effectively inhibited atherosclerotic plaque progression and improved aortic vasodilatory function in ApoE-/- rats fed a high-fat diet. At the cellular level, ISOF enhanced cell viability and proliferation of MAECs compromised by ox-LDL. These phenotypic improvements were attributed to the inhibitory effect of ISOF on endothelial-to-mesenchymal transition (EndMT); ISOF increased the expression of endothelial markers such as CD31 and E-cadherin while decreasing the expression of mesenchymal markers, including N-cadherin and α-SMA. Mechanistically, and consistent with the results of proteomic analysis, ISOF markedly inhibited the TGF-β/Smad3 signaling pathway both in vivo and in vitro. Furthermore, the expression levels of adenylyl cyclase 5 (AC5) were significantly higher than those of other AC isoforms in rat aorta. ISOF might upregulate the expression of AC5, subsequently activating downstream protein kinase A (PKA) but not cAMP-activated exchange protein-1 (EPAC1). However, when AC5 was silenced, the effects of ISOF on EndMT and the cAMP/PKA/TGF-β pathway were effectively abolished.The study showed that ISOF effectively combats atherosclerosis by inhibiting EndMT through the regulation of the AC5-dependent cAMP/PKA/TGF-β axis. This finding suggests a potential promising therapeutic strategy for treating atherosclerosis.Copyright © 2025. Published by Elsevier GmbH.