Nicorandil, a hybrid nitrate and potassium channel opener, protects ischemic myocardium by opening mitochondrial ATP-sensitive potassium (mitoKATP) channels. We previously reported that nitric oxide (NO) also opened KATP channels in rabbit hearts by a protein kinase G (PKG) mechanism. This study examined whether the NO-donor property of nicorandil also contributes to opening mitoKATP channels through PKG. MitoKATP channel opening was monitored in adult rabbit cardiomyocytes by measuring reactive oxygen species (ROS) production with reduced MitoTracker Red. Nicorandil increased ROS production in a dose-dependent manner. The selective mitoKATP channel inhibitor 5-hydroxydecanoate (200 μM) completely blocked ROS production by nicorandil at all doses. The PKG inhibitor 8-bromoguanosine-3′, 5′-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPs, 50 μM) shifted the dose-ROS production curve rightward with an increase of the EC50 from 2.4 × 10−5 M to 6.9 × 10−5 M. Rp-8-Br-cGMPs did not affect the increase in ROS production by the selective mitoKATP channel opener diazoxide, whereas it completely blocked increased ROS production from the NO donor S-nitroso-N-acetylpenicillamine (1 μM). These results indicate that nicorandil does open mitoKATP channels indirectly via a NO-PKG signaling pathway in addition to its direct effect on the channels.