Expression of voltage-gated K⁺ (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E _m) and cytosolic free Ca²⁺concentration ([Ca²⁺]_cyt). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated [Ca²⁺]_cyt following activation of K⁺ channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by α-difluoromethylornithine (DFMO) reduced whole cell K⁺ currents [I _K(v)] through Kv channels and caused membrane depolarization, which was associated with decreases in [Ca²⁺]_cyt, RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO onI _K(v) , E _m, [Ca²⁺]_cyt, and RhoA protein and restored cell migration to normal. Elevation of [Ca²⁺]_cytinduced by the Ca²⁺ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca²⁺ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C₃ transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca²⁺-activated RhoA activity.