PP2A serine/threonine protein phosphatases are heterotrimeric complexes that have a wide range of essential physiologic functions. The B55α form of PP2A has critical roles in cell cycle regulation, mitotic exit, and the DNA damage response1-6. Its activity is modulated by additional regulatory proteins, such as ARPP197, FAM122A8, and IER59. However, the precise mechanisms underlying the modulation of PP2A activity by these proteins remain elusive. Here, we show that IER5 inhibits pTau dephosphorylation by PP2A/B55α in biochemical assays and report a cryoelectron microscopy structure of the PP2A/B55α-IER5 complex, which reveals that IER5 occludes a surface on B55α used for substrate recruitment10-12. Mutation of interface residues on IER5 interferes with recovery of B55α in co-immunoprecipitation assays and suppresses events in squamous carcinoma cells, such as KRT1 expression, that depend on inhibition of PP2A/B55α by IER59. These studies define the molecular basis for PP2A inhibition by IER5 and suggest a roadmap for selective pharmacologic modulation of PP2A/B55α complexes.