Fructose-rich diets (FRD) cause cardiac insulin resistance manifested by impairment of Akt/endothelial NO synthase (eNOS) signalling. In contrast, oestradiol (E2) activates this signalling pathway in the heart. To study the ability of E2 to revert the detrimental effect of fructose on cardiac Akt/eNOS, female rats were subjected to a FRD and ovariectomy followed with or without E2 replacement. We also analysed the effects of the FRD and E2 on cardiac extracellular signal-regulated kinase (Erk 1/2) signalling related to their role in cardiac hypertrophy development. Expression of Akt, eNOS and Erk 1/2, as well as regulatory phosphorylations of these molecules were determined. The protein expression of cardiac Akt and eNOS was not affected by the diet or E2 treatment. However, the FRD was accompanied by a decrease in Akt phosphorylation at Ser473 and Thr308, and eNOS at Ser1177, while the phosphorylation of eNOS at Thr495 was increased. E2 replacement in ovariectomised fructose-fed rats caused a reversion of the diet effect on Akt and eNOS serine phosphorylation, but mostly had no effect on threonine phosphorylation of the molecules. The FRD and E2 treatment did not influence Erk 1/2 expression and phosphorylation and heart mass as well. The data show that E2 selectively suppress the negative effects of a FRD on Akt/eNOS signalling and probably point to the different effects of E2 on kinase/phosphatase pathways responsible for phosphorylation/dephosphorylation of Akt and eNOS. Furthermore, the results suggest that the heart of females in the reproductive period is partially protected against the damaging effects of increased fructose intake.