Background & Aims: N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous tetrapeptide which has antifibrogenic effects at physiological concentrations in various tissues. AcSDKP is produced locally in the liver, however, little is known about its biological effect in this organ. We hypothesize that basal levels of endogenous AcSDKP decrease during the development of liver fibrosis and preservation of basal AcSDKP attenuates liver fibrosis. Methods: Endogenous levels of AcSDKP in the liver were measured by enzyme immunoassay after 2, 6, and 10 weeks of carbon tetrachloride (CCl 4)-induced liver fibrosis in rats. Subcutaneous osmotic pump infusion of vehicle or AcSDKP (800 μg/kg/day) was administered to CCl 4-treated rats for 8 weeks to study the effect of exogenous AcSDKP on liver fibrosis. The effect of AcSDKP on profibrogenic properties of hepatic stellate cells was studied in vitro. Results: Endogenous AcSDKP was significantly decreased in the liver of CCl4-treated rats. Chronic AcSDKP infusion preserved basal levels of AcSDKP and reduced liver injury, inflammation, fibrosis, and profibrogenic transforming growth factor-β signaling. This was demonstrated by decreased aminotransferase serum levels, CD45 positive cells, collagen accumulation, α-smooth muscle actin positivity, transforming growth factor-β1, phosphorylated Smad2/3 protein, increased bone morphogenetic protein-7, and phosphorylated Smad1/5/8. Further, AcSDKP exerts antifibrogenic effects on hepatic stellate cells (HSCs) by downregulation of HSC activation in vitro. Conclusions: Maintaining physiological levels of AcSDKP is critical in negatively regulating the development of fibrosis in chronic liver injury. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis. © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.