Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin.IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.