The development of dendritic arbors is critical in neuronal circuit formation, as dendrites are the primary sites of synaptic input. Morphologically specialized dendritic protrusions called spines represent the main postsynaptic compartment for excitatory neurotransmission. Recently, we demonstrated that chicken acidic leucine-rich epidermal growth factor (EGF) -like domain-containing brain protein/neuroglycan C (CALEB/NGC), a neural member of the EGF family, mediates dendritic tree and spine complexity but that the signaling pathways in the respective processes differ. For a more detailed characterization of these signal transduction pathways, we performed a yeast two-hybrid screen to identify proteins that interact with CALEB/NGC. Our results show that B56, a regulatory subunit of protein phosphatase 2A, interacts with CALEB/NGC and inhibits CALEB/NGC-mediated dendritic branching but not spine formation. Binding of B56 to CALEB/NGC was confirmed by several biochemical and immunocytochemical assays. Using affinity chromatography and mass spectrometry, we demonstrate that the whole protein phosphatase 2A trimer, including structural and catalytic subunits, binds to CALEB/NGC via B56. We show that CALEB/NGC induces the phosphorylation of Akt in dendrites. Previously described to interfere with Akt signaling, B56 inhibits Akt phosphorylation and Akt-dependent dendritic branching but not Akt-independent spine formation induced by CALEB/NGC. Our results contribute to a better understanding of signaling specificity leading to neuronal process differentiation in sequential developmental events.—Brandt, N., Franke, K., Johannes, S., Buck, F., Harder, S., Hassel, B., Nitsch, R., Schumacher, S. B56, a regulatory subunit of protein phosphatase 2A, interacts with CALEB/NGC and inhibits CALEB/NGC-mediated dendritic branching.