Rho-guanine nucleotide dissociation inhibitor-β (RhoGDIβ), a regulator for Rho GTPases, is implicated in cancer cell progression. We reported that C-terminal truncated RhoGDIβ (ΔC(166–201)-RhoGDIβ) promoted metastasis through activating Rac1 signaling pathway in ras-transformed fibroblast cells. To better understand the mechanism of Rac1 activation by ΔC(166–201)-RhoGDIβ during metastasis, the amount of GTP-bound Rac1 was measured as the activation level of Rac1 in cells expressing various mutant RhoGDIβ with sequential C-terminal deletions. Three C-terminal hydrophobic amino acid residues (Trp191, Leu193, and Ile195) supposed to interact with isoprenyl groups of Rac1, was indispensable for a proper regulation of Rac1 activation/inhibition. Deletion of this region led RhoGDIβ to continuously associate with GTP-bound Rac1, provoking constitutive activation of Rac1. Thus, impaired interaction of RhoGDIβ with Rac1 isoprenyl groups possibly makes RhoGDIβ function as a positive regulator for Rac1 during metastasis.