To suppress enzymatic reduction of nitroxyl group of spin probes, this study designed two new nitroxyl probes, 4-hydroxy and 4-oxopiperidine-N-oxyls having 4'-hydroxyspirocyclohexyl groups at the 2- and 6-positions of the piperidine ring (hydroxy-DICPO and oxo-DICPO, respectively). The decay of the EPR signal of these probes in mouse liver homogenates was significantly suppressed compared with that of 4-hydroxy- and 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (hydroxy-TEMPO and oxo-TEMPO, respectively), although hydroxy-DICPO and oxo-DICPO showed no difference in the reactivities with ascorbic acid. While both hydroxy- and oxo-DICPO reacted with hydroxyl radicals, only hydoxy-DICPO lost its EPR signal by the reaction with superoxide anion radical in the presence of cysteine. This feature is similar to that observed for hydroxy- and oxo-TEMPO. These results suggest that the introduction of spirocyclohexyl groups to nitroxyl spin probes is effective for protecting the nitroxyl group against enzymatic reduction without changing the characteristics of the reaction with oxygen radicals.