Despite the development of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, there is an urgent need for efficient drugs to treat infected patients. An attractive drug target is the human transmembrane protease serine 2 (TMPRSS2) because of its vital role in the viral infection mechanism of SARS-CoV-2 by activation of the virus spike protein (S protein). Having in mind that the information derived from quantum mechanics/molecular mechanics (QM/MM) studies could be an important tool in the design of transition-state (TS) analogue inhibitors, we resorted to adiabatic QM/MM calculations to determine the mechanism of the first step (acylation) of proteolytic cleavage of the S protein with atomistic details. Acylation occurred in two stages: (i) proton transfer from Ser441 to His296 concerted with the nucleophilic attack of Ser441 to the substrate's P1-Arg and (ii) proton transfer from His296 to the P1'-Ser residue concerted with the cleavage of the ArgP1-SerP1' peptide bond, with a Gibbs activation energy of 17.1 and 15.8 kcal mol-1, relative to the reactant. An oxyanion hole composed of two hydrogen bonds stabilized the rate-limiting TS by 8 kcal mol-1. An analysis of the TMPRSS2 interactions with the high-energy, short-lived tetrahedral intermediate highlighted the limitations of current clinical inhibitors and pointed out specific ways to develop higher-affinity TS analogue inhibitors. The results support the development of more efficient drugs against SARS-CoV-2 using a human target, free from resistance development.