The substantial rise in multidrug-resistant bacterial infections is a current global imperative. Cumulative efforts to characterize antimicrobial resistance in bacteria has demonstrated the spread of six families of multidrug efflux pumps, of which resistance-nodulation-cell division (RND) is the major mechanism of multidrug resistance in Gram-negative bacteria. RND is composed of a tripartite protein assembly and confers resistance to a range of unrelated compounds. In the major enteric pathogen Campylobacter jejuni, the three protein components of RND are posttranslationally modified with N-linked glycans. The direct role of N-linked glycans in C. jejuni and other bacteria has long been elusive. Here, we present the first detailed account of the role of N-linked glycans and the link between N-glycosylation and antimicrobial resistance in C. jejuni We demonstrate the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilizing protein complexes and the promotion of protein-protein interaction, thus mediating antimicrobial resistance via enhancing multidrug efflux pump activity. This affirms that glycosylation is critical for multidrug efflux pump assembly. We present a generalized strategy that could be used to investigate general glycosylation system in Campylobacter genus and a potential target to develop antimicrobials against multidrug-resistant pathogens.IMPORTANCE Nearly all bacterial species have at least a single glycosylation system, but the direct effects of these posttranslational protein modifications are unresolved. Glycoproteome-wide analysis of several bacterial pathogens has revealed general glycan modifications of virulence factors and protein assemblies. Using Campylobacter jejuni as a model organism, we have studied the role of general N-linked glycans in the multidrug efflux pump commonly found in Gram-negative bacteria. We show, for the first time, the direct link between N-linked glycans and multidrug efflux pump activity. At the protein level, we demonstrate that N-linked glycans play a role in enhancing protein thermostability and mediating the assembly of the multidrug efflux pump to promote antimicrobial resistance, highlighting the importance of this posttranslational modification in bacterial physiology. Similar roles for glycans are expected to be found in other Gram-negative pathogens that possess general protein glycosylation systems.