The bovine papillomavirus capsid protein L2 has no homology to known cellular proteins. We identified the interaction of bovine papillomavirus type 1 L2 with the guanine exchange factor Vav2. We determined that the interaction of L2 with Vav2 was mediated by the N-terminus of L2 and independent of the N-terminus of Vav2. L2 expression resulted in a change in the intracellular expression of Vav2 from diffuse to punctate cytoplasmic pattern. Our experiments in human epithelial cells showed that L2 expression results in a loss of phosphorylation of cofilin, an actin depolymerizing factor through the inactivation of Vav2 and RhoA. Cofilin and RhoA have been implicated in mediating endocytosis and in the differentiation mechanism of keratinocytes. We show that bovine papillomavirus type 1pseudovirions interact with Vav2 during infection and that infection efficiency is dependent on the RhoA activation state. Our experiments suggest that L2, through Vav2/RhoA/cofilin, may regulate endocytosis of viral particles and the mechanism of cellular proliferation and differentiation during virus production. Our data propose the Vav2-related pathway as a potential therapeutic target for papillomavirus infection and oncogenic development as has been shown for breast cancer invasiveness.