Beneficial paracrine roles of mesenchymal stem cells (MSCs) in tissue repairs promise potential therapeutic strategies against different diseases. However, key therapeutic factors secreted from MSCs and their exact molecular mechanisms of action are not well defined. We found that the cell-free secretome of umbilical cord-derived MSCs had significant anti-fibrotic activity in mouse models of liver fibrosis. Our study demonstrated that the reduction in fibrosis was achieved by perturbing hepatic stellate cell activation by directly inhibiting TGFβ/Smad-signaling. Importantly, the anti-fibrotic effects of MSC secretome were blocked in vitro and in vivo by antagonizing milk fat globule-EGF factor 8 (MFGE8) activity. Furthermore, we showed that MFGE8 was secreted by MSCs not only from umbilical cord, but also from other tissues, including teeth and bone marrow. We proved that administration of recombinant MFGE8 protein alone had a significant anti-fibrotic effect using two different models of liver fibrosis. Additionally, the MFGE8 downregulated TGFβ type I receptor expression by binding to αvβ3 integrin on HSCs. These findings uncover a potential new role of MFGE8 in modulating TGFβ-signaling and suggest that MFGE8 could serve as a novel therapeutic agent for liver fibrosis.