Astroglial cells are involved in the neuropathogenesis of several inflammatory diseases of the brain, where the activation of inflammatory mediators and cytokines plays an important role. We have previously demonstrated that ethanol up-regulates inflammatory mediators in both brain and astroglial cells. Since Rho GTPases are involved in inflammatory responses of astrocytes where loss of stress fibers takes place and RhoE/Rnd3 disorganizes the actin cytoskeleton, the aim of the present study was to investigate the implication of this protein in the stimulation of inflammatory signaling induced by ethanol. Our findings show that RhoE expression induces a decrease in both RhoA and Rac. In addition, RhoE not only induces actin cytoskeleton disorganization but it also stimulates both the IRAK/ERK/NF-kappaB pathway and the COX-2 expression associated with the inflammatory response in these cells. Our results also show that ethanol exposure induces RhoE signaling in astrocytes. Preincubation of astrocytes with GF109203X, an inhibitor of PKCs, reduces the RhoE levels and abolishes the ethanol-induced activation of IRAK, NF-kappaB and the COX-2 expression. Furthermore, RhoE overexpression restores ethanol responses in astrocytes treated with the PKCs inhibitor. Altogether, our findings suggest that this small GTPase is involved in the stimulation of the inflammatory signaling induced by ethanol in astrocytes. These findings provide new insights into the molecular mechanism involved in the inflammatory responses in astrocytes.