It has long been known that glycosylation of viral envelope proteins is essential for infectivity and affects immune recognition. Surprisingly, few clinical applications have been developed using this knowledge, and glycosylation has rarely been considered in vaccine design. However, awareness is increasing and more and more studies are addressing the need of relevant glycosylation in vaccine formulations (Li et al. 2016; Go et al. 2017). The rapidly evolving analytical methods now allow precise characterization of the location and structure of both N- and O-linked glycosylation on viruses. Likewise, the advancement of glycobiology and gene editing techniques offers ample opportunities to investigate the functional roles of glycan structures and specific sites. Unreliable prediction of O-glycosylation have discouraged site-specific analysis of O-glycosylation in viruses, but now the structural information obtained by high throughput glycoproteomic approaches as well as bioinformatic analysis of glycosylation patterns can serve as a resource for defining targets for such studies. In addition, the knowledge base can be used for addressing the role of O-glycosylation for antigenicity and immunogenicity of subunit vaccine candidates. Currently it is difficult to obtain site occupancy information at a proteome-wide scale, but soon this will be possible with optimized targeted glycoproteomics strategies. Accumulating evidence suggest that O-glysosylation of viruses play important roles in various aspects of virus biology. The advent of readily available and robust genome engineering tools provides opportunities to investigate these findings in more detail and map the structural requirements for O-glycan structures on the virus and potential interaction partners on epithelial cells by using glycoengineered cell libraries (Radhakrishnan et al. 2014; Yang et al. 2015). The challenge is, however, to apply such knowledge for clinical purposes. Since viral glycosylation is orchestrated by the capacity of the host cell, general glycosylation inhibitors are of limited use due to toxicity. It is therefore important to identify structural determinants of combinatorial self- and nonself-nature for safe targeting of viral glycans and boosting antiviral immunity.