In this report, we show that GalNAc-T3 and T7 together are sufficient to initiate multiple O-glycosylations near the furin cleavage site of SARS-CoV-2 spike protein, which inhibit furin processing of the spike protein, suppress the assembly of S protein into VLPs and inhibit the infectivity of the latest omicron variant of SARS-CoV-2 in human lung cells, providing strong evidence for the regulatory role of O-glycosylation in viral activation and infection. Here, FURIN is linked to infection.