Milk thistle is a plant that has been used medicinally for over 2000 years. It has been used for the treatment of many diseases such as cancer, liver, kidney, cardiac, brain. Their seeds are rich in silymarin compounds (especially silybin A and silybin B) and fatty acids. The purpose of this study was to extract silybin A and silybin B. from milk thistle seeds with the supercritical CO2. The effect of operating parameters such as temperature (40-80 degrees C), pressure (160-220 bar), CO2 flow rate (3, 4 and 5 mL/min) and particle size (0.3025, 0.925 and 1.2 mm) on extracted oil, silybin A and silybin B were investigated. Fatty acid composition in milk thistle seed extract was determined at optimum conditions. The results indicated that the optimal conditions were 40 degrees C temperature, 200 bar pressure, 4 mL/min CO2 flow rate and 0.3025 mm particle size. In these conditions; the amounts of oil, silybin A and silybin B were obtained 327, 2.29 and 1.92 mg/g milk thistle seeds respectively. For the fatty acids obtained by supercritical CO2 extraction, the most abundant compounds were palmitic acid (8.15%); stearic acid (5.51%), oleic acid (24.10%); linoleic acid (54.97%) at optimum conditions. (C) 2015 Elsevier B.V. All rights reserved.