Immune cells migrate via actomyosin contractility mediated by myosin IIA activation, wherein the lysosomal Ragulator complex-MPRIP interaction is crucial. However, the precise mechanism underlying lysosome-mediated myosin IIA activation has not been elucidated. Here, we found that calcium efflux from the lysosomal TRPML1 channel promotes leukocyte trafficking by enhancing the interaction between the Ragulator complex and MPRIP. Disrupting the lysosome-anchoring site of Lamtor1 impaired the localization of the Ragulator complex to lysosomes, diminishing the TRPML1-mediated leukocyte migration and interaction between Lamtor1 and MPRIP. Furthermore, ouabain, a cardiac glycoside, dissociated Lamtor1 from lysosomes, inhibiting the interaction between the Ragulator complex and myosin IIA activation, thereby suppressing cell migration. Therapeutically, ouabain ameliorated the severity of MSU-induced gouty arthritis and LPS-induced lung injury in mice by inhibiting leukocyte infiltration. Overall, lysosomes facilitate the interaction between the Ragulator complex and MPRIP by supplying calcium ions through TRPML1 channels, thereby activating myosin IIA and promoting leukocyte migration.