Neuron-derived clone 77 (Nur77) is an orphan nuclear receptor with currently no known natural ligands. Here we applied a metabolomics platform for detecting protein-metabolite interactions (PMIs) to identify lipids that bind to Nur77. Using this approach, we discovered that the Nur77 ligand-binding domain (Nur77LBD) enriches unsaturated fatty acids (UFAs) in tissue lipid mixtures. The interaction of Nur77 with arachidonic acid and docosahexaenoic acid was subsequently characterized using a number of biophysical and biochemical assays. Together these data indicate that UFAs bind to Nur77LBD to cause changes in the conformation and oligomerization of the receptor. UFAs are the only endogenous lipids reported to bind to Nur77, which highlights the use of metabolomics in the discovery of novel PMIs.