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Protein phosphatase 4 (PP4) has been reported to be indispensable for cell proliferation and survival. Deletion of PP4 has been shown to induce abnormal and even lethal events in growth and development both in lower eukaryotes and in mammals. However, until now, effects of PP4 up-regulation have remained unclear.To test effects of PP4 on cell proliferation, cell cycle and morphology in HepG2 cells, it was down-regulated using PP4 siRNA or its activity was inhibited using PP4RL (a PP4 phosphatase-dead mutant) adenoviruses. Alternatively, PP4 was up-regulated using PP4 adenoviruses. Next, we used a functional proteomic approach to identify proteins that may interact with PP4. Furthermore, we performed rescue experiments to verify the possible mechanisms.To our surprise, we found that both up-regulation and inhibition of PP4 inhibited cell proliferation. Unlike PP4 inhibition, PP4 up-regulation induced prominent arrest at the prometaphase/metaphase transition by causing defects in chromosome alignment and spindle assembly. Moreover, we identified scaffold attachment factor A (SAF-A) (an important protein required for kinetochore-microtubule attachment that participates in the prometaphase/metaphase transition), to be a novel protein that interacts with PP4, using a proteomic approach. Thus, mutual regulatory mechanisms exist between PP4 and SAF-A. Interactions between PP4 and SAF-A played a role in prometaphase/metaphase transition.Our data demonstrate a novel regulatory mechanism involving PP4 in cell proliferation.