Apical-basal cell polarity must be tightly controlled for epithelial cyst and tubule formation, and these are important functional units in various epithelial organs. Polarization is achieved through the coordination of several molecules that divide cells into an apical domain and a basolateral domain, which are separated from tight and adherens junctions. Cdc42 regulates cytoskeletal organization and the tight junction protein ZO-1 at the apical margin of epithelial cell junctions. MST kinases control organ size through the regulation of cell proliferation and cell polarity. For example, MST1 relays the Rap1 signal to induce cell polarity and adhesion of lymphocytes. Our previous study showed that MST3 was involved in E-cadherin regulation and migration in MCF7 cells.In vivo, MST3 knockout mice exhibited higher ENaC expression at the apical site of renal tubules, resulting in hypertension. However, it was not clear whether MST3 was involved in cell polarity. Here, control MDCK cells, HA-MST3 and HA-MST3 kinase-dead (HA-MST3-KD) overexpressing MDCK cells were cultured in collagen or Matrigel. We found that the cysts of HA-MST3 cells were fewer and smaller than those of control MDCK cells; ZO-1 was delayed to the apical site of cysts and in cell-cell contact in the Ca2+switch assay. However, HA-MST3-KD cells exhibited multilumen cysts. Intensive F-actin stress fibers were observed in HA-MST3 cells with higher Cdc42 activity; in contrast, HA-MST3-KD cells had lower Cdc42 activity and weaker F-actin staining. In this study, we identified a new MST3 function in the establishment of cell polarity through Cdc42 regulation.