The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a global public health threat . The efficacy of several repurposed drugs has been evaluated in clinical trials . Among these drugs, a second-generation antiandrogen agent, enzalutamide, was proposed because it reduces the expression of transmembrane serine protease 2 (TMPRSS2), a key component mediating SARS-CoV-2-driven entry, in prostate cancer cells . However, definitive evidence for the therapeutic efficacy of enzalutamide in COVID-19 is lacking . Here, we evaluated the antiviral efficacy of enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and Ad-ACE2-transduced mice . Tmprss2 knockout significantly inhibited SARS-CoV-2 infection in vivo . Enzalutamide effectively inhibited SARS-CoV-2 infection in human prostate cells, however, such antiviral efficacy was lacking in human lung cells and organoids . Accordingly, enzalutamide showed no antiviral activity due to the AR-independent TMPRSS2 expression in mouse and human lung epithelial cells . Moreover, we observed distinct AR binding patterns between prostate cells and lung cells and a lack of direct binding of AR to TMPRSS2 regulatory locus in human lung cells . Thus, our findings do not support the postulated protective role of enzalutamide in treating COVID-19 through reducing TMPRSS2 expression in lung cells.