We studied markers of apoptosis and mitochondrial protection in three groups of isolated rat hearts, which underwent a) 2.5-hours perfusion without ischemia (Sham), b) 30-min ischemia (I) plus 2-hours reperfusion (R), or c) postconditioning (PostC) protocol, respectively. PostC consisted in 5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min I, at the onset of R. The marker levels of pro- and anti-apoptotic factors were studied in the cytosolic fraction (CF). The anti-apoptotic Bcl-2 protein levels resulted higher in Sham and PostC than in the I/R group (26.35 0.13 and 23.95 0.25 vs 5.64 0.07 arbitrary units). Also protective Pim-1 level resulted lower in I/R group with respect to Sham and PostC groups (20.02 0.20 vs 35.02 0.13 and 28.84 0.31). The levels of pro-apoptotic cytochrome C and caspase-3 cytosolic proteins were high in the CF of I/R group and low in the CF of Sham and PostC groups. Heat shock protein 60 (HSP60), Connexin-43 (CX43) and GSK3b were also studied in mitochondrial fraction (MF) where HSP60 level was higher in Sham and PostC than in I/R group (33.8 0.58 and 27.9 0.4 vs 1.6 0.04). Yet, in the CF, HSP60 level was higher in the I/R group than Sham and PostC groups (13.14 0.79 vs 1.25 0.09 and 1.27 0.09). In CF, CX43 levels were higher in Sham and PostC groups than I/R group (24.00 0.03 and 28.33 0.19 vs 19.33 0.19). Moreover, in MF phospho-CX43 level was higher in the I/R than Sham group (7.84 0.13 vs 0.71 0.19, p < 0.001). PostC treatment induced a reduction of phospho-CX43 with respect to I/R group (5.55 0.21 vs 7.84 0.13). While in CF, total GSK3b was more expressed in I/R group than the other two groups (10.29 0.80 vs 4.58 0.12 and 4.50 0.14) in MF, phospho-GSK3b was more expressed in PostC group with respect to Sham and I/R groups (5.98 0.10 vs 4.72 0.13 and 4.50 0.12). Electron microscopy confirmed that I/R induced marked damages of cristae and of mitochondrial membranes, which were drastically reduced by PostC. Data suggest that mitochondrial protection and anti-apoptotic effects are central aspects of PostC protection. Protective kinases (i.e Pim-1 and phospho-GSK3b) play a pivotal role in these protective mechanisms.