The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies. Display omitted •Over 100 mutations were selected for analyses on their infectivity and antigenicity•The dominant D614G itself and combined with other mutations are more infectious•Ablation of both N331 and N343 glycosylation at RBD drastically reduced infectivity•Ten mutations such as N234Q, L452R, A475V, and V483A was markedly resistant to some mAbs Eighty natural variants and 26 glycosylation spike mutants of SARS-CoV-2 were analyzed in terms of infectivity and antigenicity using high throughput pseudovirus assay in conjunction with neutralizing antibodies.