AbstractO‐glycosylation is an ancient yet underappreciated protein posttranslational modification, on which many bacteria and viruses heavily rely to perform critical biological functions involved in numerous infectious diseases or even cancer. But due to the innate complexity of O‐glycosylation, research techniques have been limited to study its exact role in viral attachment and entry, assembly and exit, spreading in the host cells, and the innate and adaptive immunity of the host. Recently, the advent of many newly developed methodologies (e.g., mass spectrometry, chemical biology tools, and molecular dynamics simulations) has renewed and rekindled the interest in viral‐related O‐glycosylation in both viral proteins and host cells, which is further fueled by the COVID‐19 pandemic. In this review, we summarize recent advances in viral‐related O‐glycosylation, with a particular emphasis on the mucin‐type O‐linked α‐N‐acetylgalactosamine (O‐GalNAc) on viral proteins and the intracellular O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) modifications on host proteins. We hope to provide valuable insights into the development of antiviral reagents or vaccines for better prevention or treatment of infectious diseases.