Abstract Background Fibroblast/myofibroblast transdifferentiation following acute cardiac injury (MI) is a major mechanism of scar formation and adverse remodeling regulated by TGFβ/SMAD3 signaling. Myofibroblasts represent phenotypically modulated cells characterized by α-smooth muscle actin (α-SMA) and constitute the main source of extracellular matrix proteins such as collagen type I in the healing infarct wound. Moreover, the recruitment of a specific monocyte/macrophages subset (Ly6Chigh and Ly6Clow) control the healing process in the infarcted wound. The forkhead transcription factor FOXO3a has recently been shown to inhibit cardiac hypertrophy by different stressors and is involved in the immune response following injury. Purpose We hypothesized that FOXO3a, a key regulator of cell differentiation, cell cycle and size as well as the stress response, might inhibit matricellular remodeling following myocardial infarction by regulating transdifferentiation of fibroblasts into myofibroblasts. Moreover, we hypothesized that Foxo3a−/− mice favor the recruitment of monocytes of the type Ly6Clow leading to an anti-inflammatory profile and thus the remodeling after injury. Methods Acute myocardial infarction was induced in FOXO3−/− and WT mice (FVB background) by permanent LAD ligation. The myocardial infarction size was determined via ECHO. Myofibroblast trans-differentiation and fibrosis markers were analyzed by QT-PCR and protein analysis. The differentiation of macrophages studied via FACS. IP/IF and Western blotting were used to test for a direct interaction between FOXO3a and SMAD3 in vitro. Results FOXO3a−/− mice showed significantly higher survival rates compared to WT littermates. Myocardial inflammation (Mac-1, LFA, CD3, CD45), injury markers such as Troponin T were similar in both groups on day 4 post-MI. Myocardial expression of alpha smooth muscle actin (ASMA) and Collagen1A1 (Col1A1), as well as numbers of cardiac CD11b+/F4–80+/Ly6clow macrophages was significantly enhanced in FOXO3a−/− mice 15 days post infarction. Moreover, Foxo3a−/− mice showed larger fibrotic areas following MI. Mechanistically, immunoprecipitation showed direct interaction of FOXO3a with SMAD3a that was enhanced following activation of the transcription factor leading to diminished SMAD3 downstream gene expression. Conclusion Our results indicate that FOXO3a acts as a direct inhibitor of TGF-β regulated myofibroblast differentiation and matrix remodeling via FOXO3a-SMAD3 interaction following MI. Moreover, a Foxo3a deficiency might lead to the recruitment of monocytes type Ly6Clow leading to an anti-inflammatory profile in the infarct wound. Therefore, FOXO3a collectively regulates cardiac hypertrophy and fibrosis/scar formation following MI. Thus, targeting the FOXO3a-SMAD3 signaling axis might be of future therapeutic interest. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): German Resaerch Foundation