To study the regulation of embryonic development by Rho, we microinjected Clostridium botulinum C3-exoenzyme (C3) into zebrafish embryos. We found that C3 inhibited cytokinesis during early cleavages. C3 inhibition appeared to be specific on RhoA, since the constitutively active RhoA could partially rescued the C3-induced defects. Distributions of actin and the cleavage furrow associated beta-catenin were disrupted by C3. Belbbistatin, a myosin II inhibitor, also caused blastomeres disintegration. It suggested that Rho mediates cytokinesis via cleavage furrow protein assembly and actomyosin ring constriction. Furthermore, C3 blocked cellular movements during epiboly and gastrulation as evident by the impairment on no tail and goosecoid expression in blastoderm front runner cells and the dorsal lip of blastopore, respectively. Y-27632, an antagonist of Rho-associated kinase (ROK/ROCK), had the similar inhibitory effects on zebrafish development as the C3 treatments. Taken together, these results suggest that Rho mediates cleavage furrow protein assembly during cytokinesis and cellular migration during epiboly and gastrulation via a ROK/ROCK-dependent pathway.