Mst1 (mammalian sterile20-like kinase 1) is activated by both caspase-mediated cleavage and phosphorylation during apoptosis induced by Fas/CD95, staurosporine or genotoxic agents. However, the physiological regulator of Mst1 has not been identified. In the present study, we demonstrate that Mst1 is regulated by the tumor suppressor RASSF1A which regulates mitosis by inhibiting APC/C and induces a prolonged G2/M arrest followed by apoptosis. We show that Mst1 interacts with RASSF1A and they colocalize to microtubules throughout the cell cycle. While the activity of Mst1 is inhibited by direct addition of purified RASSF1A in vitro, overexpression of RASSF1A augments the kinase activity of Mst1 in mammalian cells, suggesting that in vivo regulation of Mst1 by RASSF1A is more complex than simple association between them. Moreover, both apoptosis and Mst1 activation are significantly suppressed in RASSF1A-depleted cells, suggesting that RASSF1A induces the activation of Mst1 during apoptosis. Overall, we propose that RASSF1A is required for Mst1 activation and contributes to the apoptotic function of Mst1 by regulating the Mst1 kinase activity in vivo.