SARS-CoV-2 spike (S) glycoprotein mediates viral entry and membrane fusion. Its cleavage at S1/S2 and S2' sites during the biosynthesis in virus-producer cells and viral entry are critical for viral infection and transmission. In contrast, the biological significance of the junction region between both cleavage sites on S protein synthesis and function is less understood. By analyzing the conservation and structure of S protein, we find that intra-chain contacts formed by the conserved tyrosine (Y) residue 756 (Y756) with three α helices contribute to the spike's conformational stability. When Y756 is mutated to an amino acid residue that can provide hydrogen bonds, S protein could be expressed as a cleaved form, but not vice versa. Also, the L753 mutation linked to the Y756 hydrogen bond prevents the S protein from being cleaved. Y756 and L753 mutations alter S protein subcellular localization. Importantly, Y756 and L753 mutations are demonstrated to reduce the infectivity of the SARS-CoV-2 pseudoviruses by interfering with the incorporation of S protein into pseudovirus particles and causing the pseudovirus to lose its sensitivity to neutralizing antibodies. Furthermore, both mutations affect the assembly and production of SARS-CoV-2 virus-like particles in cell culture. Altogether, our findings reveal for the first time a critical role for the conserved L753-LQ-Y756 motif between S1/S2 and S2' cleavage sites in S proteins synthesis and processing as well as virus assembly and infection. IMPORTANCE The continuous emergence of SARS-CoV-2 virus variants such as delta or lambda lineage caused the continuation of the epidemic and challenged the effectiveness of these existing vaccines. Logically, the spike (S) protein mutation has attracted much concern. However, the key amino acids in S protein for its structure and function are still not very clear. Here, we discovered for the first time that the conserved residues Y756 and L753 at the junction between S1/S2 and S2' sites are very important as the S2' cleavage site R815 for the synthesis and processing of S protein such as protease cleavage, and then the mutations severely interfered with the incorporation of S protein into pseudotyped virus particles and SARS-CoV-2 virus-like particles. Consequently, we delineate the novel potential target for the design of broad-spectrum antiviral drugs in the future, especially in the emergence of SARS-CoV-2 variants.