Cephalopod molluscs are exceptional athleticinvertebrates that have evolved a high performancecardio-branchial system comprised of three hearts. Ascellular powerhouses, mitochondria have central roles inaerobic heart function. This study tested the acute andchronic impacts of thermal challenges on acclimation andevolution of mitochondria from branchial and systemichearts. Specimens from temperate and subtropicalpopulations of the common cuttlefish Sepia officinaliswere acclimated to 11°C, 16°C and 21°C. Permeabilisedheart fibres were then used to assess mitochondrialfunction using high-resolution respirometry, and asubstrate-inhibitor protocol; followed by measurementsof mitochondrial content and glycolytic enzyme activity.Mitochondria showed varying temperature dependentfuel preferences with proline being a favoured substrateat high temperatures and carbohydrates at lowtemperatures. In addition, apparent contributions of theelectron transport system relative to the phosphorylationsystem diminished with rising temperatures in temperatecuttlefish. This indicates very efficient mitochondrialcoupling at thermal limits, where the effects of thermalacclimation of mitochondrial function were most evident.Smaller hearts with higher oxidative capacities wereapparent in the subtropical specimens, while largerhearts with lower respiratory capacities were favoured intemperate specimens. Apparent fuel preferences for aminoacids and carbohydrates as well as glycolytic enzymeactivities also differed substantially between populations.In conclusion, this study provides evidence that Sepiaofficinalis hearts exhibit various bioenergetic adaptationsin response to acute, seasonal, and evolutionaryexposures to different habitat temperatures. Thisunderlines the high adaptive plasticity of the commoncuttlefish to cope with a broad range of thermalchallenges.