Mantle cell lymphoma (MCL) is a relatively rare subtype of B-cell non-Hodgkin lymphoma (NHL) that has a poor prognosis despite recent advances in immunochemotherapy and molecular targeted therapeutics against NHL. Therefore, the development of a new therapeutic strategy for MCL is urgently needed. In this study, we show for the first time that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), an oncogenic serine-threonine protein kinase, is commonly expressed in its phosphorylated active form in patient-derived tumor cells of various types of B-cell NHL cells, including diffuse large B-cell lymphoma, follicular lymphoma, and MCL. Blockade of PDPK1 activity by small-molecule inhibitors specific for PDPK1 (BX-912 and GSK2334470) or by RNA interference exerted antiproliferative effects in all four MCL-derived cell lines examined and these growth-inhibitory effects were mediated by both induction of apoptosis and G2/M cell cycle blockade. In addition, blockade of PDPK1 led to inactivation of its downstream effector kinase RSK2, but not AKT, suggesting the importance of the PDPK1/RSK2 signaling pathway in the proliferation and survival of MCL cells. Finally, when combined with anticancer agents, including genotoxic agents, a proteasome inhibitor, and a BH3 mimetic in vitro, the PDPK1 inhibitor BX-912 showed additive growth-inhibitory effects against MCL-derived cell lines in most settings. In particular, the combination of BX-912 and ABT-263, a BH3 mimetic, resulted in the enhancement of the induction of apoptosis. In conclusion, our results suggest that PDPK1 is a potential novel therapeutic target in MCL and indicate that clinical development of PDPK1-targeted therapy for MCL is desirable.