The rat is an important alternative for studying human pathology owing to certain similarities to humans. Glycomic studies on rat serum have revealed that variations in the N-glycans of glycoproteins correlated with disease progression, which is consistent with the findings in human serum. Therefore, we comprehensively characterized the rat serum N-glycome using microfluidic chip-LC-ESI-QTOF MS and MS/MS techniques. In total, 282 N-glycans, including isomers, were identified. This study is the first to present comprehensive profiling of N-glycans containing O-acetylated sialic acid, among which 27 N-glycans are novel. In addition, the co-existence of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) in a single N-glycan ('mixed' N-glycan) was detected and represents a new type of N-glycan in rat serum. The existence of O-acetylated sialic acid is the characteristic feature of rat serum that distinguishes it from mouse and human sera. Comparisons between the rat, mouse, and human serum glycomes revealed that the rat glycome is more similar to that of human sera than to that of mouse sera. Our findings highlight the similarities between the glycomic profile of rat and human sera and provided important selection criteria for choosing an appropriate animal model for pathological and pharmacological studies.