A supercritical fluid carbon dioxide (SF-CO2) extraction method was used to obtain the optimum process for extracting yellow horn seed oil. The anti-fatigue and antioxidant properties of the extracted oil were investigated through animal experiments. The optimum process conditions for SF-CO2extraction of the yellow horn oil were 40 MPa, 50 °C and 120 min, with an extraction yield of 31.61%. The high-dose group of yellow horn oil could significantly increase the weight-bearing swimming time, the hepatic glycogen (HG) content and decrease the lactic acid (LA) content and blood urea nitrogen (BUN) content (p< 0.05) in mice. Moreover, it improved the antioxidant ability by reducing the malondialdehyde (MDA) content (p< 0.01) and raising the glutathione reductase (GR) content and superoxide dismutase (SOD) content (p< 0.05) in mice. Yellow horn oil has the effects of being an anti-fatigue and antioxidant substance, which provides a basis for its further utilization and development.