The altered N-glycosylation of glycoproteins has been suggested to play an important role in the behavior of malignant cells. Using glycomics technology, we attempted to determine the specific and detailed N-glycan profile for hepatocellular carcinoma (HCC) and investigate the prognostic capabilities. From 1999 to 2011, 369 patients underwent primary curative hepatectomy in our facility and were followed up for a median of 60.7 months. As normal controls, 26 living Japanese related liver transplantation donors were selected not infected by hepatitis B and C virus. Their mean age was 40.0 and 15 (57.7%) were male. We used a glycoblotting method to purify N-glycans from preoperative blood samples from this cohort (10 lL serum) which were then identified and quantified using mass spectrometry (MS). Correlations between the N-glycan levels and the clinicopathologic characteristics and outcomes for these patients were evaluated. Our analysis of the relative areas of all the sugar peaks identified by MS, totaling 67 N-glycans, revealed that a proportion had higher relative areas in the HCC cases compared with the normal controls. Fourteen of these molecules had an area under the curve of greater than 0.80. Analysis of the correlation between these 14 N-glycans and surgical outcomes by univariate and multivariate analysis identified G2890 (m/z value, 2890.052) as a significant recurrence factor and G3560 (m/z value, 3560.295) as a significant prognostic factor. G2890 and G3560 were found to be strongly correlated with tumor number, size, and vascular invasion. Conclusion: Quantitative glycoblotting based on whole serum N-glycan profiling is an effective approach to screening for new biomarkers. The G2890 and G3560 N-glycans determined by tumor glycomics appear to be promising biomarkers for malignant behavior in HCCs. (HEPATOLOGY 2013;57:2314-2325 H epatocellular carcinoma (HCC) is a common and fatal malignancy with a worldwide occurrence.1 Liver resection has shown the highest level of control among the local treatments for HCC and is associated with a good survival rate.2,3 However, the recurrence rates for HCC are still high even when a curative hepatectomy is performed. 4 Many factors associated with the prognosis and recurrence of HCC have now been reported. Vascular invasion of the portal vein and/or hepatic vein and tumor differentiation are important factors affecting survival and recurrence in HCC cases after a hepatectomy. 5,6 However, microvascular invasion and differentiation can only be detected by pathological examination just after a hepatectomy, and cannot be diagnosed preoperatively, and thus cannot be identified preoperatively either. Hence, the serum biomarkers alpha-fetoprotein (AFP) and protein induced by vitamin K absence-II (PIVKA-II) are used as prognostic markers 7,8 and also as surrogate markers for microvascular invasion and tumor differentiation.9,10 AFP is associated with grade differentiation, 11 whereas PIVKA-II is related to vascularAbbreviations: AFP, alpha-fetoprotein; AFP-L3, lens cul...