Mammalian nuclear Dbf2-related (NDR) kinases (LATS1 and 2, NDR1 and 2) play a role in cell proliferation, apoptosis and morphological changes. These kinases are regulated by mammalian sterile 20-like kinases (MSTs) and Mps one binder (MOB) 1. Okadaic acid (OA), which activates MST2, facilitates the complex formation of MOB1, MST2 and NDR1 in HEK293FT cells. The in vitro biochemical study demonstrates the phosphorylation of MOB1 by MST2. The phosphorylated MOB1 alone is capable to partially activate NDR1 in vitro, but MST2 is also required for the full activation. The knockdown of MOB1 or MST2 abolishes the OA-induced NDR1 activation in HEK293FT cells. Among MOB1 mutants, in which each serine or threonine residue is replaced with alanine, MOB1 T74A and T181A mutants fail to activate NDR1. Thr74, but not Thr181, is phosphorylated by MST2 in vitro, although MOB1 is also phosphorylated by MST2 at other site(s). The interaction of MOB1 T74A with NDR1 is barely enhanced by OA treatment. These findings indicate that the phosphorylation of MOB1 at Thr74 by MST2 is essential to make a complex of MOB1, MST2 and NDR1, and to fully activate NDR1.