Purpose: The changes of glycosylation of different IgG subclass in colorectal cancer (CRC) were rarely investigated. The authors aimed to use a simple and high-throughput analytical method to explore the changes of subclass-specific IgG glycosylation in CRC, and to find the specific glyco-biomarkers for early detection of this disease. Experimental design: Serum samples from 71 cancer patients and 22 benign patients with 50 age- and sex-matched healthy controls were collected from two independent cohorts. Subclass-specific IgG glycosylation was profiled by MALDI-MSfollowed by the structural identification through MALDI-MS/MS. The exported MS data was automatically and rapidly processed by the self-developed MATLAB code. Results: Statistical analysis suggested the significantly decreased galactosylation and remarkably increased agalactosylation of IgG1 or IgG2 in the malignant transformation of CRC, which enables the differentiation between cancer patients and healthy controls. The changes of glycan features were elucidated by the exploration of individual glycopeptides, showing the biantennary fucosylated glycan without galactose (H3N4F1) or with two galactose (H5N4F1) of IgG1 and IgG2 could distinguish cancer group from both benign and control groups. Conclusions and clinical relevance: Through the simple and high-throughput procedures, this study revealed the important role of IgG glycopeptides in the premature pathology of CRC.