The Kaposi’s sarcoma herpesvirus (KSHV) open reading frame 74 encodes a G protein-coupled receptor (GPCR) for chemokines. Exogenous expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi’s sarcoma. We show here that expression of KSHV-GPCR in transfected cells resulted in constitutive transactivation of nuclear factor kappa-B (NF-kB) and secretion of IL-8, and this response involves activation of Galpha(13) and RhoA. The induced expression of a NF-kB luciferase reporter was partially reduced by pertussis toxin and the Gbetagamma scavenger transducin, and enhanced by co-expression of Galpha(13) and to a lesser extent, Galpha(q). These results indicate coupling of KSHV-GPCR to multiple G proteins for NF-kB activation. Expression of KSHV-GPCR led to stress fiber formation in NIH 3T3 cells. To examine the involvement of the Galpha(13)¡VRhoA pathway in KSHV-GPCR-mediated NF-kB activation, HeLa cells were transfected with KSHV-GPCR alone and in combination with the regulator of G protein signaling (RGS) from p115RhoGEF or a dominant negative RhoA (T19N). Both constructs, as well as the C3 exoenzyme from Clostritium botulinum, partially reduced NF-kB activation by KSHV-GPCR, and by a constitutively active Galpha(13)(Q226L). KSHV-GPCR-induced NF-kB activation is accompanied by increased secretion of IL-8, a function mimicked by the activated Galpha(13) but not by an activated Galpha(q)(209L). These results suggest coupling of KSHV-GPCR to the Galpha(13)¡VRhoA pathway in addition to other G proteins.