Galectin-3 (Gal-3) is secreted by activated macrophages. In hypertension, Gal-3 is a marker for hypertrophic hearts prone to develop heart failure (HF). Gal-3 infused in pericardial sac leads to cardiac inflammation, remodeling and dysfunction. N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP), a natural occurring tetrapeptide, prevents and reverses inflammation and collagen deposition in the heart in hypertension and HF post myocardial infarction. In the present study, we hypothesize that Ac-SDKP prevents Gal-3-induced cardiac inflammation, remodeling, and dysfunction and these effects are mediated by the TGF/Smad3 signaling pathway. Adult male rats were divided into four groups and received the following intrapericardial infusion for 4 weeks: 1) vehicle (saline, n = 8), 2) Ac-SDKP (800 µg/kg/day, n = 8), 3) Gal-3 (12 µg/day, n = 7), and 4) Ac-SDKP + Gal-3 (n = 7). Left ventricular ejection fraction (LVEF), cardiac output (CO) and transmitral velocity were measured by echocardiography; inflammatory cell infiltration, cardiomyocyte hypertrophy, and collagen deposition in the heart by histological and immunohistochemical staining; and TGF- expression and Smad3 phosphorylation by Western blot. We found that in the LV, Gal-3: 1) enhanced macrophage and mast cell infiltration, increased cardiac interstitial and perivascular fibrosis and causes cardiac hypertrophy; 2) Increased TGF- expression and Smad3 phosphorylation; and 3) decreased -dP/dt response to isoproterenol challenge, E/A ratio and LVEF. Ac-SDKP partially or completely prevented these effects. We conclude that Ac-SDKP prevents Gal-3 induced cardiac inflammation, fibrosis, hypertrophy and dysfunction possibly via inhibition of the TGF-/Smad3 signaling pathway.