Development of strategies to prevent herpes simplex virus (HSV) infection requires knowledge of cellular pathways harnessed by the virus for invasion. This study demonstrates that HSV induces rapid phosphorylation of focal adhesion kinase (FAK) in several human target cells and that phosphorylation is important for entry post-binding. Nuclear transport of the viral tegument protein VP16, transport of viral capsids to the nuclear pore, and downstream events (including expression of immediate-early genes and viral plaque formation) were substantially reduced in cells transfected with dominant-negative mutants of FAK or small interfering RNA designed to inhibit FAK expression. These observations were substantiated using mouse embryonic fibroblast cells derived from embryonic FAK-deficient mice. Infection was reduced by >90% in knockout cells relative to control cells and was further reduced if the knockout cells were transfected with small interfering RNA targeting proline-rich tyrosine kinase-2, which was also phosphorylated in response to HSV. The knockout cells were permissive for viral binding, and virus triggered an intracellular calcium response, but nuclear transport was inhibited. Together, these results support a novel model for invasion that implicates FAK phosphorylation as important for delivery of viral capsids to the nuclear pore.