Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear. Using osteosarcoma cells, we found that in addition to its known localizations on microtubules and the plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and focal adhesions. Cells depleted of MARK proteins demonstrated that MARK2 promotes phosphorylation of both myosin II and the myosin phosphatase targeting subunit MYPT1 to synergistically drive myosin II contractility and stress fiber formation in cells. Studies with isolated proteins showed that MARK2 directly phosphorylates myosin II regulatory light chain, while its effects on MYPT1 phosphorylation are indirect. Using a mutant lacking the membrane-binding domain, we found that membrane association is required for focal adhesion targeting of MARK2, where it specifically enhances cell protrusion by promoting FAK phosphorylation and formation of focal adhesions oriented in the direction of migration to mediate directionally persistent cell motility. Together, our results define MARK2 as a master regulator of the actomyosin and microtubule cytoskeletal systems and focal adhesions to mediate directional cancer cell migration. •MARK2 associates with the actomyosin cytoskeleton•MARK2 promotes phosphorylation of MRLC and MYPT1 to drive contractility•MARK2 associates with focal adhesions via its membrane-binding domain•MARK2 promotes FAK activation and focal adhesion formation and orientation Pasapera et al. show that the microtubule regulatory and polarity protein MARK2 associates with actomyosin and promotes phosphorylation of myosin II regulatory light chain and MYPT1 to drive contractility. MARK2 also associates with focal adhesions (FAs) and promotes FAK activation and FA formation/orientation to drive directional cell migration.