In this study, we established an experimental assay system monitoring ACE2- and TMPRSS2-dependent SARS-CoV-2 S protein-mediated membrane fusion, in 293FT and Calu-3 cells and found that nafamostat mesylate potently blocked SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-type-dependent manner. Here, PROS1 is linked to viral infectious disease.