AbstractMetazoan proteomes contain many paralogous proteins that have evolved distinct functions. The Ena/VASP family of actin regulators consists of three members that share an EVH1 interaction domain with a 100% conserved binding site. A proteome-wide screen revealed ciliary protein PCARE as a high-affinity ligand for ENAH EVH1. Here we report the surprising observation that PCARE is ~100-fold specific for ENAH over paralogs VASP and EVL and can selectively bind and inhibit ENAH-dependent adhesion in cells. Specificity arises from a mechanism whereby PCARE stabilizes a conformation of the ENAH EVH1 domain that is inaccessible to family members VASP and EVL. Structure-based modeling rapidly identified seven residues distributed throughout EVL that are sufficient to differentiate binding by ENAH vs. EVL. By exploiting the ENAH-specific conformation, we rationally designed the tightest and most selective ENAH binder to date. Our work uncovers a conformational mechanism of interaction specificity that distinguishes highly similar paralogs and establishes tools for dissecting specific Ena/VASP functions in processes including cancer cell invasion.