Recombinant lentivirus is widely used as a gene transfer vehicle for gene therapy and tissue engineering due to high transduction efficiency and its ability to infect non-dividing cells and stem cells in vitro and in vivo. However, the interactions between lentivirus and the target cells during transduction process are only partially understood. Here we show that transduction with recombinant lentivirus activated the c-jun-N-terminal kinase (JNK) pathway by phosphorylating JNK in primary epidermal cells and several epithelial cell lines. Blocking JNK phosphorylation with chemical inhibitors and siRNA decreased gene transfer in a dose dependent manner. Similar results were observed with actively growing as well as non-proliferating cells indicating that JNK may affect gene transfer independent of cell cycle status of target cells. In addition, we found that inhibition of JNK affected early events in the gene transfer process. Specifically, activation of JNK was necessary of internalization of viral particles in the cell cytoplasm as evidenced by measurements of internalized viral proteins using western blots and ELISA. Immunostaining results further indicated that JNK affected viral internalization may through regulating filopodia formation of the target cells. These results suggest activation of JNK may be necessary for lentivirus to gain entry into the target cells.