Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria natural infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that CD81 is required on hepatocytes for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 belongs to the tetraspanin superfamily of transmembrane proteins. By interacting with each other and with other transmembrane proteins, tetraspanins may play a role in the lateral organization of membrane proteins. In this study, we investigated the role of the two major molecular partners of CD81 in hepatocytic cells, CD9P-1/EWI-F and EWI-2, two transmembrane proteins belonging to a novel subfamily of immunoglobulin proteins. We show that CD9P-1 silencing increases the host cell susceptibility to P. yoelii sporozoite infection, whereas EWI-2 knock-down has no effect. Conversely, overexpression of CD9P-1 but not EWI-2 partially inhibits infection. Using CD81 and CD9P-1 chimeric molecules, we demonstrate the role of transmembrane regions in CD81-CD9P-1 interactions. Importantly, a CD9P-1 chimera that no longer associates with CD81 does not affect infection. Based on these data, we conclude that CD9P-1 acts as a negative regulator of P. yoelii infection by interacting with CD81 and regulating its function.