Update information. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, has caused a worldwide pandemic since its discovery at the end of 2019. The spike (S) protein of SARS-CoV-2 is responsible for the host cell entry and the critical targets of the immune system. Consequently, S protein has been extensively used in antibody and vaccine studies. Here, we identified a total of 23 N-glycosites, including N334 in the noncanonical consensus motif N-X-C, on the full-length wild-type S protein protomer. Furthermore, 55 O-glycosylation sites were identified, and this uncovered a pattern of O-glycosylation near N-glycosites, which we named the “O-follow-N” rule, revealed by site-specific mutagenesis. The comprehensive mapping of the S protein O- and N-glycosylation landscape provides further information for studying the mechanisms underlying viral pathogenesis and immune evasion.