The host–guest interaction of nitroxide radicals with water-soluble pillar[n]arenes was studied for the first time by electron paramagnetic resonance spectroscopy and NMR spectroscopy. Our results showed that this interaction strongly depended on the 4-substituents of nitroxides and the cavity size of pillar[n]arenes. The host–guest interaction with water-soluble pillar[6]arene WP6 effectively increased the thermodynamic and kinetic stability of nitroxide radical 4-AT toward ascorbic acid, thus expanding its potential biomedical applications.