Non-muscle myosin II is an actin-based motor protein essential to cell motility, division, migration, adhesion and polarity. This myosin forms a hexameric complex comprised of two heavy chains (NMHC-II), two essential light chains, and two regulatory light chains (RLC). In vertebrates, there are three NMHC-II isoforms (NMHC-IIA, NMHC-IIB, and NMHC-IIC), which exhibit distinct patterns of expression in cells and tissues. Regulation of NMHC-II activity occurs through RLC and HC phosphorylation. RLCs are phosphorylated at Thr-18 and Ser-19, which activates myosin II motor activity and promotes filament stability. By contrast, PKC phosphorylation of Ser-1/Ser-2 and Thr-9 in RLC may decrease activated myosin II interaction with actin. Several kinases phosphorylation NMHC-II isoforms directly. TRPM7 phosphorylates Thr-1800, Ser-1803, and Ser-1808, which reduces NMHC-IIA incorporation into the actin cytoskeleton. PKC phosphorylates NMHC-IIA on Ser-1916 and NMHC-IIB on multiple tailpiece serines leading to inhibition of filament assembly. Casein kinase II phosphorylates NMHC-IIA on Ser-1943 and increases disassembly of NMHC-IIA filaments.