The aim of our study was to characterize effect of ischemic preconditioning (PC) mediated by PI3/K/Akt signaling pathway during ischemia/reperfusion (I/R) injury in Langendorff-isolated rat hearts under conditions of acute hyperglycemia (HG) simulated by perfusion with Krebs-Henseleit solution with elevated glucose concentration (22 mmol/L). PC was induced by two cycles of 5-min coronary occlusion/5-min reperfusion prior to 30 min coronary occlusion/120 min reperfusion. The severity of I/R injury was characterized by determination of the size of infarction (IS, expressed in % of area at risk size) and the amount of heart-type fatty acid binding protein (h-FABP, a marker of cell injury) released from the hearts to the effluent. Samples for Western blot (WB) analysis were taken from hearts before and after exposure to  global 30 min ischemia/40 min reperfusion. Activity of PI3/K/Akt was expressed as P-Akt/Akt ratio. Levels of BAX were measured after I/R. Higher IS and h-FABP amount released from preconditioned hearts against controls in HG conditions point out to negative effect of PC on heart resistance against I/R injury unlike in conditions of standard glucose (11 mmol/L, “normoglycemia NG”) perfusion, where differences in IS and released h-FABP were inverted and prosurvival PC effect was confirmed. Significant increase of P-Akt/Akt prior to I/R and its decrease together with reduced BAX after I/R in preconditioned hearts under NG indicates PI3K/Akt benefitial effect and probable negative feedback regulation which prevents chronic deleterious PI3K/Akt activity. Only insignificant P-Akt/Akt increase without its decrease and insignificant changes of BAX at the end of I/R in PC hearts under HG indicate attenuated PI3K/Akt signaling, failure of feedback mechanisms and antiapoptotic activity.