AbstractRho GTPases are master regulators of the eukaryotic cytoskeleton. The activation of Rho GTPases is governed by Rho guanine nucleotide exchange factors (GEFs). Three RhoGEF isoforms are produced by the gene ARHGEF25; p63RhoGEF⁵⁸⁰, GEFT and a recently discovered longer isoform of 619 amino acids (p63RhoGEF⁶¹⁹). The subcellular distribution of p63RhoGEF⁵⁸⁰ and p63RhoGEF⁶¹⁹ is strikingly different in unstimulated cells, p63RhoGEF⁵⁸⁰ is located at the plasma membrane and p63RhoGEF⁶¹⁹ is confined to the cytoplasm. Interestingly, we find that both P63RhoGEF⁵⁸⁰ and p63RhoGEF⁶¹⁹ activate RhoGTPases to a similar extent after stimulation of Gαq coupled GPCRs. Furthermore, we show that p63RhoGEF⁶¹⁹ relocates to the plasma membrane upon activation of Gαq coupled GPCRs, resembling the well-known activation mechanism of RhoGEFs activated by Gα_12/13. Synthetic recruitment of p63RhoGEF⁶¹⁹ to the plasma membrane increases RhoGEF activity towards RhoA, but full activation requires allosteric activation via Gαq. Together, these findings reveal a dual role for Gαq in RhoGEF activation, as it both recruits and allosterically activates cytosolic ARHGEF25 isoforms.