This chapter describes signaling pathways, stimulated by the P2Y nucleotide receptor (P2YR), that regulate cellular processes dependent on actin cytoskeleton dynamics in glioma C6 cells. P2YR coupled with G-proteins, in response to ATP or UTP, regulates the level of iphosphatidylinositol-4,5-bisphosphate (PIP) which modulates a variety of actin binding proteins and is involved in calcium response and activates Rac1 and RhoA proteins. The RhoA/ROCK signaling pathway plays an important role in contractile force generation needed for the assembly of stress fibers, focal adhesions and for tail retraction during cell migration. Blocking of this pathway by a specific Rho-kinase inhibitor induces changes in F-actin organization and cell shape and decreases the level of phosphorylated myosin II and cofilin. In glioma C6 cells these changes are reversed after UTP stimulation of P2YR. Signaling pathways responsible for this compensation are calcium signaling which regulates MLC kinase activation via calmodulin, and the Rac1/PAK/LIMK cascade. Stimulation of the Rac1 mediated pathway via G proteins needs additional interaction between αβ integrins and P2YRs. Calcium free medium, or growing of the cells in suspension, prevents Gα activation by P2Y receptors. Rac1 activation is necessary for cofilin phosphorylation as well as integrin activation needed for focal complexes formation and stabilization of lamellipodium. Inhibition of positive Rac1 regulation prevents glioma C6 cells from recovery of control cell like morphology.