ABSTRACTIn eukaryotes, glycosylation plays a role in proteome stability, protein quality control, and modulating protein function; however, similar studies in bacteria are lacking. Here, we investigate the roles of general protein glycosylation systems in bacteria using the enteropathogenCampylobacter jejunias a well-defined example. By using a quantitative proteomic strategy, we were able to monitor changes in theC. jejuniproteome when glycosylation is disrupted. We demonstrate that inC. jejuni, N-glycosylation is essential to maintain proteome stability and protein quality control. These findings guided us to investigate the role ofN-glycosylation in modulating bacterial cellular activities. In glycosylation-deficientC. jejuni, the multidrug efflux pump and electron transport pathways were significantly impaired. We demonstrate thatin vivo, fully glycosylation-deficientC. jejunibacteria were unable to colonize its natural avian host. These results provide the first evidence of a link between proteome stability and complex functions via a bacterial general glycosylation system.IMPORTANCEAdvances in genomics and mass spectrometry have revealed several types of glycosylation systems in bacteria. However, why bacterial proteins are modified remains poorly defined. Here, we investigated the role of generalN-linked glycosylation in a major food poisoning bacterium,Campylobacter jejuni. The aim of this study is to delineate the direct and indirect effects caused by disrupting this posttranslational modification. To achieve this, we employed a quantitative proteomic strategy to monitor alterations in theC. jejuniproteome. Our quantitative proteomic results linked general proteinN-glycosylation to maintaining proteome stability. Functional analyses revealed novel roles for bacterialN-glycosylation in modulating multidrug efflux pump, enhancing nitrate reduction activity, and promoting host-microbe interaction. This work provides insights on the importance of general glycosylation in proteins in maintaining bacterial physiology, thus expanding our knowledge of the emergence of posttranslational modification in bacteria.