We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking PLC- and PKC. Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKCa in a PS/DG-independent manner. Moreover, NIH3T3 cells expressing the 4CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to PMA, compared to parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEF) of Ahnak knockout (Ahnak-/-) mouse. Membrane translocation of PKCa and phosphorylation of Raf in response to PMA or PDGF was decreased in Ahnak null MEF cells compared to wild type MEF. Several lines of evidence suggest that PKCa activity is regulated through association with protein phosphatase 2A (PP2A). Co-immunoprecipitation assay indicated that the association of PKCa with PP2A was disrupted in NIH3T3 cells expressing 4CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A.