C1q/tumor necrosis factor-related protein-6 (CTRP6) is a newly identified adiponectin paralog with modulation effects on metabolism and inflammation. However, the cardiovascular function of CTRP6 remains unknown. This study aimed to determine its role in cardiac fibrosis and explore the possible mechanism. Myocardial infarction (MI) was induced by left anterior descending coronary artery ligation in rats. CTRP6 was mainly expressed in the cytoplasm of adult rat cardiomyocytes and significantly decreased in the border and infarct zones post-MI. Adenovirus-mediated CTRP6 delivery improved cardiac function, attenuated cardiac hypertrophy, alleviated cardiac fibrosis, and inhibited myofibroblast differentiation as well as the expression of collagen I, collagen III, and connective tissue growth factor post-MI. In cultured adult rat cardiac fibroblasts (CFs), exogenous or cardiomyocyte-secreted CTRP6 inhibited, whereas knockdown of CTRP6 facilitated transforming growth factor-β1 (TGF-β1)-induced expression of α-smooth muscle actin, smooth muscle 22α, and profibrotic molecules. CTRP6 had no effect on CFs proliferation but attenuated CFs migration induced by TGF-β1. CTRP6 increased the phosphorylation of AMP-activated protein kinase (AMPK) and Akt in CFs and post-MI hearts. Pretreatment with adenine 9-β-d-arabinofuranoside (AraA), an AMPK inhibitor, or LY294002, a phosphatidylinositol-3-kinase (PI3 K) inhibitor, abolished the protective effect of CTRP6 on TGF-β1-induced profibrotic response. Furthermore, CTRP6 had no effect on TGF-β1-induced Smad3 phosphorylation and nuclear translocation, whereas significantly decreased TGF-β1-induced RhoA activation and myocardin-related transcription factor-A (MRTF-A) nuclear translocation, and these effects were blocked by AMPK or Akt inhibition. In conclusion, CTRP6 attenuates cardiac fibrosis via inhibiting myofibroblast differentiation. AMPK and Akt activation are responsible for the CTRP6-mediated anti-fibrotic effect by targeting RhoA/MRTF-A pathway.