Abstract Brassinosteroids may importantly affect safflower (Carthamus tinctorius L.) growthand quality under stress. The oil responses of three spring safflower genotypes (Goldasht, Faraman and Sina) affected by the exogenous application of 24- epibrassinolide (EBL, 0.1 μM) under drought stress (85 (I1), 65 (I2) and 45 % (I3) of the field capacity) were investigated in 2016. The stress decreased oil percentage and yield, fatty acid yield and stomatal conductance, whereas supplying sufficient water did not alter fatty acid content. EBL increased oil and fatty acid percentage (except palmitic acid), and stomatal conductance. Foliar EBL application led to enhancement of oleic (15.95 %), linolenic (22.50 %), linoleic (4.26 %) and stearic acid (29.6 %) compared with control. The hormonal treatment also enhanced oil and fatty acid yield in I1 and I2. The desaturation efficiency and unsaturation indexes indicated that EBL can affect safflower response under the stress by desaturating 18:2 fatty acids to 18:3 fatty acids. Safflower oil content and fatty acids, as a function of plant genotype, can be enhanced under drought stress using EBL. The presented research can also further enhance our understanding related to lipid metabolism in safflower affected by EBL under drought stress. Such results are of nutritional, economic, industrial and environmental significance.