•N-glycosylation deficient mutants were more susceptible against Pst DC3000 than wild type plants.•COS induces resistance to Pst DC3000 in N-glycosylation deficient mutants.•COS regulates N-glycosylation via a non-canonical pattern compared with Pst DC3000. Roles of protein N-glycosylation in chitosan oligosaccharide (COS) induced resistance were investigated in the present study. Results demonstrated that N-glycosylation deficient Arabidopsis mutants (stt3a and ManI) were more susceptible against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) than wild type (WT) plants. Surprisingly, in stt3a and ManI, COS-induced resistance to Pst DC3000 was mostly intact, and the up-regulation effect on SA- and JA-mediated signalling pathways also similar like WT. Nucleotide sugars accumulation and N-glycosylation related genes expression were differently regulated after COS treatment. Global glycomics analysis quantified 157 N-glycan isomers, and 56.7, 50.3 and 47.1 % of them were significantly changed in COS, mock + Pst, and COS + Pst treated plants, respectively. Moreover, COS pretreatment could reverse the effect of Pst DC3000 on many N-glycans, suggesting that COS regulates protein N-glycosylation via a non-canonical pattern compared with plant defense, which may contribute to its obvious disease control effect when N-glycosylation impairment occurs.