Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and usually develops in patients with liver cirrhosis (LC). Biomarkers that discriminate HCC from LC are important but are limited. In the present study, an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS)-based metabonomics approach was used to characterize serum profiles from HCC (n = 82), LC (n = 48), and healthy subjects (n = 90), and the accuracy of UPLC-MS profiles and alpha-fetoprotein (AFP) levels were compared for their use in HCC diagnosis. By multivariate data and receiver operating characteristic curves analysis, metabolic profiles were capable of discriminating not only patients from the controls but also HCC from LC with 100% sensitivity and specificity. Thirteen potential biomarkers were identified and suggested that there were significant disturbances of key metabolic pathways, such as organic acids, phospholipids, fatty acids, bile acids, and gut flora metabolism, in HCC patients. Canavaninosuccinate was first identified as a metabolite that exhibited a significant decrease in LC and an increase in HCC. In addition, glycochenodeoxycholic acid was suggested to be an important indicator for HCC diagnosis and disease prognosis. UPLC-MS signatures, alone or in combination with AFP levels, could be an efficient and convenient tool for early diagnosis and screening of HCC in high-risk populations. This paper was published in the Journal of Proteome Research.