Omega-3 polyunsaturated fatty acids (PUFA) provide protection against myocardial damage in ischemia-reperfusion. However, the mechanisms that provide cardioprotection are not fully understood. In this study, we investigated the effect of docosahexaenoic acid (DHA), a member of omega -3 PUFA, on mitochondrial respiration parameters and the role of mitochondrial calcium-dependent potassium channels of large conductance (ВКСа) in the implementation of these effects. Using the patch-clamp method, it was shown that functional ВКСа channels are expressed in the inner mitochondrial membrane of cardiac cells and their activity increases with the addition of DHA. We investigated the role of mitochondrial ВКСа channels in the regulation of mitochondrial respiratory processes. In experiments with isolated mitochondria from rat hearts, we showed that DHA prevented an increase in the respiratory rate of mitochondria in the V4 state and a decrease in the respiratory control elicited by addition of 10 μM Ca2+. Qualitatively the same effect was caused by NS1619, the ВКСа opener. In the presence of 10 μM Ca2+, the ВКСа channel inhibitor paxilin (1 μM) prevented the protective effect of DHA and NS1619 on the parameters of respiratory control. We conclude that mitochondrial ВКСа channels are involved in the implementation of the effects of DHA on mitochondrial respiration.