PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. Only a limited number of PP2A-regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site-specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. Strikingly, we find that B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A-B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A-B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.