COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still a threat to millions of lives worldwide. Although SARS-CoV-2 vaccines have been approved to reduce the severity and death associated with COVID-19, the number of SARS-CoV-2-infected cases still remains high, especially with the appearance of various mutant strains such as P. 1.351 and P. 1.617 (also known as South Africa strain and India strain, respectively), which may reduce the efficacy of vaccine protection. There is an urgent need to develop effective antiviral agents to treat COVID-19 patients, especially with those infected with SARS-CoV-2 variants of concern. The goal of our study was to evaluate the SARS-CoV-2 antiviral activities of Food and Drug Administration (FDA)-approved drugs, which could accelerate the development of novel therapies for COVID-19. We tested tamoxifen and clomiphene which were also used to screen antiviral agents against flavivirus. On Vero and Caco-2 cells, we first measured the cytotoxicity of the two drugs and determined that their median cytotoxic concentrations (CC50s) in vitro were much higher than our experimental range (Supplementary Fig. S1a-d). Moreover, we analyzed the antiviral activities of the two drugs and found that both tamoxifen and clomiphene had effective antiviral activities. The median effective concentrations (EC50s) of tamoxifen and clomiphene on Vero cells were 1.634 and 0.3213 μM, respectively (Fig. 1 a). The two drugs also suppressed SARS-CoV-2 infection in Caco-2 cells with a dose-dependent manner (Supplementary Fig. S1e, f). These results showed that tamoxifen and clomiphene strongly antagonized SARS-CoV-2 infection in vitro. In addition to the EC50s calculated according to SARS-CoV-2 RNA copies in cell supernatants, we also measured the spike (S) protein levels of SARS-CoV-2 in infected cells under drug treatments. Vero cells were pretreated with tamoxifen or clomiphene of different doses, and then infected with SARS-CoV-2. Vero cells were continued to be treated with drugs after infection. SARS-CoV-2 S protein was used to test viral protein production by western blot. The results suggested that tamoxifen and clomiphene reduced SARS-CoV-2 S protein production, especially the accumulation of the S2 fragment (Fig. 1 b, c). SARS-CoV-2 S protein was also examined under tamoxifen and clomiphene treatments by immunofluorescence (Fig. 1 d, e). The results showed that the two drugs reduced SARS-CoV-2 protein production in a dose-dependent manner, which further illustrated the antiviral activities of tamoxifen and clomiphene against SARS-CoV-2 infection. To understand the mechanism responsible for the antiviral activities of tamoxifen and clomiphene against SARS-CoV-2 infection, we first measured the transcriptome of Huh7 cells which were either untreated or pretreated with tamoxifen or clomiphene before SARS-CoV-2 infection to determine the effects of these drugs on SARS-CoV-2-induced gene expression profiles. The transcriptome results showed that SARS-CoV-2 infection increased the expressions of interferon (IFN) and acute inflammatory response genes, but these inductions were suppressed by tamoxifen and clomiphene treatments (Supplementary Fig. S2a, b). To further determine whether tamoxifen and clomiphene directly suppressed IFN and inflammation response genes or indirectly affected IFN and inflammation responses through inhibiting SARS-CoV-2 infection, we also analyzed the effect of tamoxifen and clomiphene on poly (I: C)-induced IFN response genes. A549 cells were pretreated with tamoxifen and clomiphene before …