Tetraspanins are integral transmembrane proteins organized in microdomains displaying specific and direct interactions with other tetraspanins and molecular partners. Among them, CD81 has been implicated in a variety of physiological and pathological processes. CD81 also plays a crucial role in pathogen entry into host cells, including hepatitis C virus (HCV) entry into hepatocytes. HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV entry into hepatocytes is a complex process that requires the coordinated interaction of viral and host factors for the initiation of infection, including CD81, scavenger receptor BI, claudin-1, occludin, membrane-bound host cell kinases, Niemann-Pick C1 Like 1, Harvey rat sarcoma viral oncogene homolog (HRas), CD63 and transferrin receptor 1. Furthermore, recent data in HCV model systems have demonstrated that targeting critical components of tetraspanins and associated cell membrane proteins open new avenues to prevent and treat viral infection.