In this study, an orthogonal array design OA₉ (3⁴) was employed to optimise the conditions of supercritical carbon dioxide (SC-CO₂) extraction of Pinus koraiensis nut oil. The effects of pressure, temperature and extraction time on the oil yield were investigated. Next, the fatty acid composition of the oil was examined by gas chromatography-mass spectrometry (GC-MS). The in vivo antioxidant activity of the oil was determined by estimating the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and the content of malondialdehyde (MDA) in rats fed with a high-fat diet. The results showed that extraction pressure and time were the main variables that influenced the oil yields. The optimal conditions with which to obtain highest yield of oil were determined to be 5760.83 psi, 50°C and 3.0 h (extraction yield was 458.5 g kg⁻¹); nine compounds, constituting about 99.98% of the total oil, were identified. The most abundant polyunsaturated fatty acids identified in the oil, linoleic acid and α-linolenic acid, constituted 41.79% and 15.62% of the oil, respectively. Moreover, the results on their antioxidant activities showed that the oil could improve the activities of SOD, GSH-Px and T-AOC, and reduce the content of MDA significantly, in the serum. These results indicate that P. koraiensis nut oil obtained by SC-CO₂ extraction had excellent antioxidant activities.