Nur77 is a transcription factor belonging to the NR4A subfamily of nuclear hormone receptors. Upon induction, Nur77 modulates the expression of its target genes and controls a variety of biological and pathophysiological processes. Prior research that revealed a structurally atypical ligand-binding domain (LBD) and failed to locate an endogenous ligand had led to a classification of Nur77 as an orphan receptor. However, several more recent studies indicate that small synthetic molecules and unsaturated fatty acids can bind to Nur77. Discovery of additional endogenous ligands will facilitate our understanding of the receptor's functions and regulatory mechanisms. Our data have identified prostaglandin A2 (PGA2), a cyclopentenone prostaglandin (PG), as such a ligand. Cyclopentenone PGs exert their biological effects primarily by forming protein adducts via the characteristic electrophilic β-carbon(s) located in their cyclopentenone rings. Our data show that PGA2 induces Nur77 transcriptional activity by forming a covalent adduct between its endocyclic β-carbon, C9, and Cys566 in the receptor's LBD. The importance of this endocyclic β-carbon was substantiated by the failure of PGs without such electrophilic properties to react with Nur77. Calculated chemical properties and data from reactive molecular dynamic simulations, intrinsic reaction co-ordinate modeling, and covalent molecular docking also corroborate the selectivity of PGA2's C9 β-carbon towards Nur77's Cys. In summary, our molecular, chemical, and structural characterization of the PGA2–Nur77 interaction provides the first evidence that PGA2 is an endogenous Nur77 agonist.