Abstract: TMPRSS2 plays a crucial role in facilitating the entry of both the influenza virus and the SARS-CoV-2 coronavirus into host cells. Recent studies have identified a guanine-rich sequence in the proximal promoter region of the TMPRSS2 gene, which can form G-quadruplex structures (TMPRSS2-G4s) that are potential targets for small molecules to inhibit TMPRSS2 expression. However, the structural details of the major TMPRSS2-G4 and its complex with small molecules remain unknown, hindering the development of antiviral drugs targeting TMPRSS2-G-quadruplexes (G4s). This study reports the first high-resolution nuclear magnetic resonance (NMR) solution structure of the major TMPRSS2-G4, which consists of a three-tetrad core parallel-stranded G4. Both 3′ and 5′ flanking regions form well-defined capping structures stabilized by multiple hydrogen bonds. Importantly, we found that berberine, an antiviral alkaloid, strongly binds to the major TMPRSS2-G4 and determined its binding complex structure with TMPRSS2-G4 at a 2∶1 binding stoichiometry. Each berberine molecule recruits an adjacent flanking residue, forming a coplanar structure superimposed on two outer G-tetrads. Moreover, we demonstrated that the major TMPRSS2-G4 can stably form within a longer deoxyribonucleic acid (DNA) context and be targeted by small molecules to inhibit DNA polymerase activity. Overall, this study provides structural insights into the recognition mechanism of small molecules by the major TMPRSS2-G4 and may facilitate the development of novel antiviral therapeutics targeting TMPRSS2-G4.