Objective. Met⁵-enkephalin (ME)-induced cardioprotection occurs via epidermal growth factor receptor (EGFR) transactivation with subsequent activation of phosphatidyl inositol-3-kinase (PI3K). In the present study, we investigated whether there is a gender difference in ME-elicited PI3K signaling. Methods. Neonatal murine cardiomyocytes were isolated by collagenase digestion, and subjected to 90 min hypoxia and 180 min reoxygenation at 37°C (n=5-7 replicates). PI3K / Akt signaling was interrogated using pharmacologic inhibitors and small interfering RNA (siRNA). Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. Data are presented as mean ± SEM. Results. There was not a gender differences in basal content of total Akt. ME (100 µM) elicited comparable protection in both genders. Wortmannin and the non-selective Akt-inhibitor IV completely abolished ME-induced protection in male cardiomyocytes but only attenuated protection in female cardiomyocytes. Isoform-selective knock-down of Akt in males with siRNA against Akt1/2 completely abolished ME-induced cardioprotection whereas siRNA against Akt3 only attenuated protection ~ 40%. In contrast, in females siRNAs against Akt1/2 attenuated and Akt3 eliminated ME-induced cardioprotection. Conclusion. (1) There is not a gender difference in the degree of ME-induced protection, and (2) there is a gender difference in cardioprotective signaling pathways after administration of ME: ME-induced cardioprotection in males primarily utilizes a PI3K — Akt1/2 pathway, and in females primarily utilizes a PI3K - Akt3 pathway. Incomplete loss of protection in females following blockade of PI3K suggests additional factors may facilitate the maintenance or function of activated Akt.