Honey bees Apis mellifera L. are one of the most studied insect species due to their economic importance. The interest in studying honey bees chiefly stems from the recent rapid decrease in their world population, which has become a problem of food security. Nevertheless, there are no systemic studies on the properties of the mitochondria of honey bee flight muscles. We conducted a research of the mitochondria of the flight muscles of A. mellifera L. The influence of various organic substrates on mitochondrial respiration in the presence or absence of adenosine diphosphate (ADP) was investigated. We demonstrated that pyruvate is the optimal substrate for the coupled respiration. A combination of pyruvate and glutamate is required for the maximal respiration rate. We also show that succinate oxidation does not support the oxidative phosphorylation and the generation of membrane potential. We also studied the production of reactive oxygen species by isolated mitochondria. The greatest production of H O (as a percentage of the rate of oxygen consumed) in the absence of ADP was observed during the respiration supported by α-glycerophosphate, malate, and a combination of malate with another NAD-linked substrate. We showed that honey bee flight muscle mitochondria are unable to uptake Ca -ions. We also show that bee mitochondria are able to oxidize the respiration substrates effectively at the temperature of 50°С compared to Bombus terrestris mitochondria, which were more adapted to lower temperatures.