Abstract This proposal is aimed to develop a method to synthesize structurally defined heparan sulfated oligosaccharides as therapeutic agents. In the current funding cycle, we propose to study the role of heparan sulfate in dampening inflammatory responses. This project is a collaborative effort of three research groups, including Dr. Jian Liu (University of North Carolina), Dr. Rafal Pawlinski (University of North Carolina), and Dr. Ding Xu (State University of New York at Buffalo). Acetaminophen overdose causes hepatocyte cell damage that triggers inflammatory responses for tissue repairing. However, if this response overreacts, it causes to collateral damages to the liver issues, leading to acute liver injury. Escalated liver injury leads to the failure of heart and lung, and ultimately death. We discover a structurally homogeneous heparan sulfate octadecasaccharide (18-mer) displays strong protection effect for the liver injury induced by acetaminophen in a murine model. Three specific aims are proposed to understand the details behind the 18-mer’s protection effect. Aim 1 is to determine the targets for the effect of 18-mer. Aim 2 is to determine the structural selectivity of heparan sulfate for the hepatoprotection effect using microarray analysis. Aim 3 is to determine the efficacy for the delayed treatment for acetaminophen-induced liver injury with a goal to widen the therapeutic window. Because uncontrolled inflammation is a major contributor to many diseases, results from our studies will advance anti-inflammatory drug development focusing heparan sulfate molecules.