Normal functions of mitochondria are required for physiological dynamics of cells, while their dysfunction contributes to development of various disorders including those of immune system. Here we demonstrate that exposure of mast cells to ragweed pollen extract increases production of H₂O₂ via mitochondrial respiratory complex III. These mitochondrial ROS (mtROS) enhance secretion of histamine and serotonin from mast cells, but not enzymes such as β-hexosaminidase, independently from FcɛRI-generated stimuli. The release of biogenic amines is associated with inhibition of secretory granules’ H⁺-ATPase activity, activation of PKC-δ and microtubule-dependent motility, and it is independent from intracellular free Ca²⁺ levels. To asses differences from IgE-mediated mast cell degranulation we show that mtROS decrease antigen-triggered β-hexosaminidase release, while they are synergistic with antigen-induced IL-4 production in sensitized cells. Taken together, these data indicate that mitochondrial dysfunction can act independently from adaptive immunity, as well as augments Th2-type responses. Pharmacological maintenance of physiological mitochondrial function could have clinical benefits in prevention and treatment of allergic diseases.