Acute-on-chronic liver failure (ACLF) is a syndrome with high short-term mortality. Rapidly occurring or exacerbated thrombocytopenia which is closely associated with adverse clinical outcomes has been frequently observed in ACLF patients. Novel approaches are needed to clarify how thrombocytopenia occurs and how platelet function is altered in ACLF patients. Using phosphorylated proteomic approaches with circulating platelets from five pairs of ACLF patients, we discovered that altered proteins might compromise regulation of actin cytoskeleton in circulating platelets from ACLF patients ceased within 28 days compared with those who survived. To our knowledge, this study is the first to examine the phosphoproteomic profile of circulating platelets in patients with liver failure. This dataset will be helpful for further elucidation of the mechanisms underlying thrombocytopenia and its potential pathogenic roles in ACLF.