RhoGDIbeta, a Rho GDP dissociation inhibitor, induced hypertrophic growth and cell migration in a cultured cardiomyoblast cell line, H9c2. We demonstrated that RhoGDIbeta plays a previously undefined role in regulating Rac1 expression through transcription to induce hypertrophic growth and cell migration and that these functions are blocked by the expression of a dominant-negative form of Rac1. We also demonstrated that knockdown of RhoGDIbeta expression by RNA interference blocked RhoGDIbeta-induced Rac1 expression and cell migration. We demonstrated that the co-expression of ZAK and RhoGDIbeta in cells resulted in an inhibition in the activity of ZAK to induce ANF expression. Knockdown of ZAK expression in ZAK-RhoGDIbeta-expressing cells by ZAK-specific RNA interference restored the activities of RhoGDIbeta.