Though heart is one of the most energy requiring organs and depends on a perfect match of energy supply with high and fluctuating energy demand, the role of AMP-activated protein kinase (AMPK) in this organ and potential sex differences are still not entirely clear. In this study, we compare some aspects of cardiac AMPK signaling between male and female mice in the context of a cardiomyocyte-specific, inducible AMPK α1/α2 KO (10 weeks after KO induction). We found that already in WT mice, the expression of AMPK α protein was higher in males than in females, an effect observed for both α isoforms, but more pronounced for AMPK α2. As a consequence, also the P-AMPK signal was higher in WT males. Cardiac mitochondria from male WT animals had higher respiratory rates with substrates of complex I, higher total capacity of electron transport chain estimated by uncoupled respiration and higher activity of complex IV. Upon AMPK α1/α2 KO, the reduction of mitochondrial respiration with substrates of complex I and of activity of complex IV was more marked in the hearts of male animals. Interestingly, respiration with fatty-acid linked substrate (palmitoylcarnitine) was significantly affected only in females. Finally, global analysis of protein kinase activities in the heart (PamGene kinase profiling) indicated sex-related differences present already in the signaling network of WT animals, but also emerging upon AMPK α1/α2 KO. Our study supports the presence of sex-specific differences in AMPK signaling. - MTS, LK and RI contributed equally to this work.