Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic, represents a serious threat to public health. The spike (S) glycoprotein of SARS-CoV-2 mediates viral entry into host cells and is heavily glycosylated. In this study, we systemically analyzed the roles of 22 putative N-linked glycans in SARS-CoV-2 S protein expression, membrane fusion, viral entry, and stability. Using α-glycosidase inhibitors, castanospermine and NB-DNJ, we confirmed that disrupting the N-linked glycosylation process blocked the maturation of the S protein, leading to impairment of S protein-mediated membrane fusion. Single substitution of each of the 22 N-linked glycosylation sites with glutamine revealed that 9 out of the 22 N-linked glycosylation sites were critical for S protein folding and maturation, resulting in reduced S protein-mediated cell-cell fusion and viral entry. Of note, N1074Q mutation markedly affected S protein stability and induced significant receptor-independent syncytium (RIS) formation in HEK293T/hACE2-KO cells, and removal of the furin-cleavage site partially compensated instability of N1074Q mutation. Although the corresponding mutation in the SARS-CoV S protein (N1056Q) did not induce RIS in HEK293T cells, the mutants N669Q and N1080Q exhibited increased fusogenic activity and induced syncytia formation in HEK293T cells. Therefore, N-glycans on the SARS-CoV and SARS-CoV-2 S2 subunits are of great importance in maintaining the prefusion state of the S protein. The study revealed the critical roles of N-glycans in S protein maturation and stability, which have implications for the design of vaccines and anti-viral strategies.