C1q/tumor necrosis factor-related protein-3 (CTRP3) is a novel adipokine with modulation effects on metabolism, inflammation, and cardiovascular system. This study aimed to investigate the effect of CTRP3 on cardiac fibrosis and its underlying mechanism. The myocardial expression of CTRP3 was significantly decreased after myocardial infarction (MI). Adenovirus-delivered CTRP3 supplement attenuated myocardial hypertrophy, improved cardiac function, inhibited interstitial fibrosis, and decreased the number of myofibroblasts post-MI. In cultured adult rat cardiac fibroblasts (CFs), CTRP3 attenuated cell proliferation; migration; and the expression of connective tissue growth factor, collagen I, and collagen III induced by transforming growth factor (TGF)-β1. Moreover, CTRP3 inhibited whereas CTRP3 small interfering RNA (siRNA) facilitated the expression of α-SMA and profibrotic molecules induced by TGF-β1. CTRP3 also attenuated TGF-β1-induced Smad3 phosphorylation, nuclear translocation, and interaction with p300. CTRP3 increased the phosphorylation of AMP-activated protein kinase (AMPK) and Akt in both rat hearts and CFs. Adenine 9-β-D-arabinofuranoside (AraA), an AMPK inhibitor, abolished the protective effect of CTRP3 against TGF-β1-induced profibrotic response and Smad3 activation. Taken together, CTRP3 attenuates cardiac fibrosis by inhibiting myofibroblast differentiation and the subsequent extracellular matrix production. AMPK is required for the anti-fibrotic effect of CTRP3 through targeting Smad3 activation and inhibiting myofibroblast differentiation. CTRP3 alleviates cardiac fibrosis in a rat post-MI model and in cardiac fibroblasts. CTRP3 inhibits fibroblast-to-myofibroblast differentiation. CTRP3 exerts anti-fibrotic effect through targeting Smad3 activation. AMPK mediates the anti-fibrotic effect of CTRP3 by inhibition of Smad3 activation.