It was recently reported that the hydroxyflavones quercetin and kaempferol bind the orphan nuclear receptor 4A1 (NR4A1, Nur77) and act as antagonists in cancer cells and tumors, and they inhibit pro-oncogenic NR4A1-regulated genes and pathways. In this study, we investigated the interactions of flavone, six hydroxyflavones, seven dihydroxyflavones, three trihydroxyflavones, two tetrahydroxyflavones, and one pentahydroxyflavone with the ligand-binding domain (LBD) of NR4A1 using direct-binding fluorescence and an isothermal titration calorimetry (ITC) assays. Flavone and the hydroxyflavones bound NR4A1, and their KDvalues ranged from 0.36 µM for 3,5,7-trihydroxyflavone (galangin) to 45.8 µM for 3′-hydroxyflavone. KDvalues determined using ITC and KDvalues for most (15/20) of the hydroxyflavones were decreased compared to those obtained using the fluorescence assay. The results of binding, transactivation and receptor–ligand modeling assays showed that KDvalues, transactivation data and docking scores for these compounds are highly variable with respect to the number and position of the hydroxyl groups on the flavone backbone structure, suggesting that hydroxyflavones are selective NR4A1 modulators. Nevertheless, the data show that hydroxyflavone-based neutraceuticals are NR4A1 ligands and that some of these compounds can now be repurposed and used to target sub-populations of patients that overexpress NR4A1.