We previously showed that polyamine depletion decreased migration, Rac activation and protein serine threonine phosphatase 2A activity. We also showed that polyamine depletion increased cortical F-actin, and decreased lamellipodia and stress fibers. In this study, we used staurosporine (STS), a potent, cell permeable, and broad-spectrum serine/threonine kinase inhibitor and studied migration. The STS concentration above 100 nM induced apoptosis. However in polyamine-depleted cells, a lower concentration of STS (5 nM) increased attachment, spreading, Rac1 activation and subsequently migration without causing apoptosis. STS-induced migration was completely prevented by Rac1 inhibitor (NSC23766) and DN-Rac1. These results imply that STS restores migration in polyamine-depleted cells through Rac1. The most important finding in this study is that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (pThr18/Ser19-MRLC) at the cell periphery, which co-localized with the thick cortical F-actin. The localization of pThr18- and pSer19-MRLC was found with stress fibers and nuclei respectively. STS decreased phosphorylation of cellular and peripheral pThr18-MRLC without any effect on nuclear pSer19-MRLC, dissolved the thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells. In control and polyamine depleted cells FAK co-localized with stress fibers and actin cortex respectively. STS reorganized FAK, paxillin, and cytoskeleton. These results suggest that polyamine depletion prevents dephosphorylation of MRLC and thereby prevents the dynamic reorganization of the actin cytoskeleton and decreases lamellipodia formation resulting in the inhibition of migration.