Protein phosphorylation is a reversible post-translational modification that is critical for the regulation of key cellular processes. It is estimated that two-thirds of all cellular proteins are phosphorylated, with more than 98% of those phosphorylation events occurring on Ser/Thr residues. Protein phosphorylation is mediated by protein kinases and reversed via dephosphorylation by protein phosphatases. Two protein phosphatases, phosphatase 1 (PP1) and Protein phosphatase 2A (PP2A), are thought to account for more than 90% of the total phosphatase activity in eukaryotic cells. PP2A is a highly conserved assortment of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). Substrate recruitment of PP2A/B55α, the most abundant PP2A holoenzyme, was thought to be mediated by charge-charge interactions between the surface of B55α and its substrates. Challenging this view, we recently discovered a conserved SLiM (Short Linear Motif) [RK]-V-x-x-[VI]-R in a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowle et al. eLife 2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55α/PP2A, and analysis of the associated proteomic datasets that aided in identifying FAM122A, which can assist in the further identification of potential substrates and cellular pathways regulated by this phosphatase. The newly identified conserved SLiM is necessary for FAM122A binding to B55α in vitro and in cells. Computational structure prediction with AlphaFold2 predicts an interaction consistent with the mutational data and supports a mechanism whereby FAM122A uses the ‘SLiM’ in the form of a short α-helix to dock to the B55α top groove. In this model, FAM122A spatially constrains substrate access by occluding the catalytic subunit with a second α-helix immediately adjacent to helix-1. Consistently, FAM122A functions as a competitive inhibitor as it prevents the binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. Ablation of FAM122A in human cell lines reduces the rate of proliferation, the progression through cell cycle transitions, and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in the attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a ‘SLiM’-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase. In agreement with these findings, ectopic expression of B55α results in the downregulation of 14-3-3σ signaling mediated by ATM and ATR as determined by pathway analysis of phosphoproteomic datasets and a reduction of ATM signaling within the total proteome. Altogether, this work has significantly expanded our understanding of the PP2A/B55 SLiM, resulting from the characterization of FAM122A, a high-affinity substrate inhibitor, and enables future interrogation of novel substrates and signaling networks regulated by PP2A/B55α. / Biomedical Sciences