Mast cells are important sentinel cells in the first line of host defense against viral and bacterial entry. SARS-CoV-2 can activate mast cells present in the respiratory tract in the initial stage of the disease. In the present study, we attempted to dissect the mechanisms underlying the interaction of the function of mast cells, especially focusing on the mast cell-derived chymase, with the cell entry of SRAS-CoV-2 by using a well-established mast cell line, rat basophil leukemia (RBL-2H3) cells. RBL-2H3 cells were infected by pseudovirons and the viral entry was evaluated. The interactions between SRAS-CoV-2 spike and mast cell-derived chymase were observed and predicated binding-site were determined by using spike-truncating variants. During the viral infection, the interaction of chymase with SRAS-CoV-2 may have both detrimental and positive impacts. Whether SRAS-CoV-2 spike is a potential substrate of chymase, and if its cleavage modulates biological properties of spike-bearing virus would be focused on in future planned study.