The conserved alphaherpesvirus serine/threonine kinase US3 causes dramatic changes in the actin cytoskeleton, consisting of actin stress fibre breakdown and protrusion formation, associated with increased virus spread. Here, we showed that US3 expression led to RhoA phosphorylation at serine 188 (S188), one of the hallmarks of suppressed RhoA signalling, and that expression of a non-phosphorylatable RhoA variant interfered with the ability of US3 to induce actin rearrangements. Furthermore, inhibition of cellular protein kinase A (PKA) eliminated the ability of US3 to induce S188 RhoA phosphorylation, pointing to a role for PKA in US3-induced RhoA phosphorylation. Hence, the US3 kinase leads to PKA-dependent S188 RhoA phosphorylation, which contributes to US3-mediated actin rearrangements. Our data suggest that US3 efficiently usurps the antagonistic RhoA and Cdc42/Rac1/p21-activated kinase signalling branches to rearrange the actin cytoskeleton.