Mammalian sterile 20-like kinase 1 (MST1) is a serine/threonine protein kinase that is activated in response to a variety of apoptotic stimuli and causes apoptosis when over-expressed in mammalian cells. The physiological regulation and cellular targets of MST1 are not well understood. Using a yeast two-hybrid system, we identified human WW45 (hWW45, also called hSav1) as an MST1-binding protein. The association between the two proteins was confirmed by immunofluorescence and co-immunoprecipitation, and hWW45 was present in both the cytoplasm and nucleus. When hWW45 alone was over-expressed, it weakly induced apoptosis. However, hWW45 augmented MST1-induced apoptosis when the two were co-expressed. Conversely, RNA interference-mediated depletion of endogenous hWW45 suppressed MST1-induced apoptosis. These results indicate that hWW45 is required to enhance MST1-mediated apoptosis in vivo and thus is a critical player in an MST1-driven cell death signaling pathway.