The COVID-19 pandemic, caused by SARS-CoV-2, leads to severe acute respiratory distress syndrome (ARDS). A crucial aspect of coronavirus infection is its unique cell entry mechanism, which involves the processing of its spike glycoprotein. The SARS-CoV-2 spike glycoprotein binds to the host receptor ACE2 and is activated by the host serine proteases Furin and TMPRSS2 through proteolytic cleavage, enabling subsequent viral entry. This study integrates structural, molecular, clinical, and computational approaches to elucidate how TMPRSS2 and Furin recognize and activate the SARS-CoV-2 spike. First, we report the structure of Furin in complex with the SARS-CoV-2 spike glycoprotein, revealing how Furin binds to and cleaves the S1/S2 region of the spike. Second, we identified TMPRSS2 cleavage sites within the S2 domain of the SARS-CoV-2 spike and determined the structure of its complex, including the catalytic triad. Furthermore, through whole-exome sequencing, we identified a genetic mutation, rs12329760 (V160M) in the TMPRSS2 gene, which is associated with a decreased infection rate in clinically diagnosed COVID-19 patients. Similarly, our analysis revealed that genetic variants/alleles in Furin modify the binding affinity for the viral spike glycoprotein, potentially explaining some of the observed diversity in infection susceptibility. These findings provide critical insights into the mechanisms and modes of action of Furin and TMPRSS2, which are hallmarks of increased viral virulence and invasion. This comprehensive understanding aids in the development of potential inhibitors targeting this infection pathway, offering promising avenues for high-quality intervention strategies against coronavirus infections.