Glycosylation is one of the most important forms of post-translational modification of proteins. Glycan influences folding and stability of proteins and their biological functions. A berrant glycosylation has been associated with many malignant tumor. Proteomic analysis methods combined with Pro-Q Emerald glycoprotein staining technology was applied to investigate the glycosylation difference of glycoprotein between normal human liver cell lines and hepatocellular carcinoma cell lines. Total proteins were extracted by using cell-splitting methods, then subjected to two-dimension electophoresis(2-DE). After that, 2-DE gel were stained with Pro-Q Emerald glycoprotein stain. Glycoprotein patterns of both cell lines were obtained. Glycosylation status of glycoproteins were quantificationally compared by using Dymention software and glycoproteins with altered glycosylation were identified with mass spectrometry. Results showed that (74±2) glycoproteins (n＝3) were detected in normal human liver cell lines whereas (78±3) glycoproteins (n＝3) were detected in human hepatocellular carcinoma cell lines. There were 31 matched glycoproteins between both cell lines. Glycoprotein patterns had distinct difference between normal human liver cells and human hepatocellular carcinoma cells. 25 glycoproteins presented glycoslytion changes in human hepatocellular carcinoma cells comparing with normal human liver cells. Among such glycoproteins, glycosylation level of 10 glycoproteins were up-regulated whereas glycosylation level of 15 glycoproteins were down-regulated and 12 of these glycoproteins had been identified by mass spectrometry. These results imply that glycosylation changes may play key roles in occurrence and development of liver cancer.