Summary CD81 is a major receptor for Hepatitis C Virus (HCV). It belongs to the tetraspanin family whose members form dynamic clusters with numerous partner proteins and with one another, forming tetraspanin‐enriched areas in the plasma membrane. In our study, we combined single‐molecule microscopy and biochemistry experiments to investigate the clustering and membrane behaviour of CD81 in the context of cells expressing EWI‐2wint, a natural inhibitor of HCV entry. Interestingly, we found that EWI‐2wint reduces the global diffusion of CD81 molecules due to a decrease of the diffusion rate of mobile CD81molecules and an increase in the proportion of confined molecules. Indeed, we demonstrated that EWI‐2wint promotes CD81 clustering and confinement in CD81‐enriched areas. In addition, we showed that EWI‐2wint influences the colocalization of CD81 with Claudin‐1 – a co‐receptor required for HCV entry. Together, our results indicate that a change in membrane partitioning of CD81 occurs in the presence of EWI‐2wint. This study gives new insights on the mechanism by which HCV enters into its target cells, namely by exploiting the dynamic properties of CD81.