Protection from a prolyl hydroxylase domain-containing enzyme (PHD) inhibitor, desferoxamine (DFO) was recently reported to be dependent on production of reactive oxygen species (ROS). Ischemic preconditioning triggers the protected state by stimulating nitric oxide (NO) production to open ATP-sensitive K⁺ channels (mitoK_ATP) generating ROS required for protection. We tested whether DFO and a second PHD inhibitor, ethyl-3,4 dihydroxybenzoate (EDHB), might have similar mechanisms. Both EDHB and DFO increased ROS generation by 50-75% (p<0.001) in isolated rabbit cardiomyocytes. This increase following EDHB exposure was blocked by either L-NAME, a NO synthase (NOS) inhibitor, ODQ, a guanylyl cyclase antagonist, and Rp-8-Br-cGMPS, a PKG blocker, thus implicating the NO pathway in EDHB's signaling. Glibenclamide, a non-selective K_ATP blocker, or 5-hydroxydecanoate, a selective mitoK_ATP antagonist, also prevented EDHB's ROS production as did blockade of mitochondrial electron transport with myxothiazol. NOS is activated by Akt. However, neither wortmannin, an inhibitor of phosphatidylinositol-3 kinase, nor Akt-inhibitor blocked EDHB-induced ROS generation, indicating EDHB initiates signaling downstream of Akt. DFO also increased ROS production, and this effect was blocked by ODQ, 5-HD, and N-(2-mercaptopropionyl)glycine (MPG), a ROS scavenger. DFO increased cardiomyocyte production of nitrite, a metabolite of NO, and this effect was blocked by an inhibitor of NOS. DFO also spared ischemic myocardium in intact hearts. This infarct-sparing effect was blocked by ODQ, L-NAME, and MPG. Hence, DFO and EDHB stimulate NO-dependent activation of PKG to open mitoK_ATP and produce ROS which act as second messengers to trigger entrance into the preconditioned state.