The vaccinia F11 protein promotes viral spread by modulating the cortical actin cytoskeleton by inhibiting RhoA signaling via an unknown mechanism. PDZ domains are widely conserved protein interaction modules whose occurrence in viral proteins is unprecedented. We found that F11 contains a central PDZ-like domain that is required to downregulate RhoA signaling and enhance viral spread. The PDZ-like domain interacts with the PDZ binding motif of the Rho GTPase-activating protein (GAP) Myosin-9A. In the absence of Myosin-9A, RhoA signaling is not inhibited, resulting in fewer actin tails and reduced virus release concomitant with less viral spread. The loss of Myosin-9A GAP activity or its ability to bind F11 also reduces actin tail formation. Furthermore, the ability of Myosin-9A to promote viral spread depends on F11 binding RhoA. Thus, F11 acts as a functional PDZ-containing scaffolding protein to inhibit RhoA signaling by binding Myosin-9A.