In the prospective Korean Cancer Prevention Study-II (KCPS-II), we investigated the application of metabolomics to differentiate subjects with incident hepatocellular carcinoma (HCC group) from subjects who remained free of cancer (control group) during a mean follow-up period of 7 years with the aim of identifying valuable metabolic biomarkers for HCC. We used baseline serum samples from 75 subjects with incident HCC and 134 age- and gender-matched cancer-free subjects. Serum metabolic profiles associated with HCC incidence were investigated via metabolomics analysis. Compared with the control group, the HCC group showed significantly higher serum levels of aspartate aminotransferase (AST), alanine aminotransferase, and -glutamyl transpeptidase. At baseline, compared with the control group, the HCC group showed significantly higher levels of 9 metabolites, including leucine, 5-hydroxyhexanoic acid, phenylalanine, tyrosine, arachidonic acid, and tauroursodeoxycholic acid (TUDCA), but lower levels of 28 metabolites, including oleamide, androsterone sulfate, L-palmitoylcarnitine, lysophosphatidic acid (LPA) 16:0, LPA 18:1, and lysophosphatidylcholines (lysoPC). Multiple linear regression revealed that the incidence of HCC was associated with the levels of tyrosine, AST, lysoPCs (16:1, 20:3), oleamide, 5-hydroxyhexanoic acid, androsterone sulfate, and TUDCA (adjusted R 2 = 0.514, P = 0.036). This study showed the clinical relevance of the dysregulation of not only branched amino acids, aromatic amino acids, and lysoPCs but also bile acid biosynthesis and linoleic acid, arachidonic acid, and fatty acid metabolism. In addition, tyrosine, AST, lysoPCs (16:1, 20:3), oleamide, 5-hydroxyhexanoic acid, androsterone sulfate, and TUDCA were identified as independent variables associated with the incidence of HCC. Cancer Prev Res; 11(5); 303–12. ©2018 AACR .