The effect of therefining process on the physicochemical properties, fatty acid composition, bioactive components and antioxidant properties of evening primrose oil(EPO) wasinvestigated. The results showed that the peroxide value, acid value and p‐anisidine value changed significantly throughout the refining process (p< 0.05). Twelve fatty acids were identified in oil samples, while theirpercentage contents were not significantly different during the refining process. Contents of tocopherol and total phenols were removedby 23.5% and 79.4%, respectively, after refining. The refining process resulted in losses of campesterolandβ‐sitosterol of approximately 16.0%and 13.8%, respectively. Moreover, squalene was detected in evening primrose oil for the first time, and its content decreased by 20.2% during the refining process. Furthermore, during the whole refining process, the DPPH free radical scavenging ability of EPO decreased by 10.6%, and the FRAP reducing capacity dropped by 56%,meanwhile, its antioxidant stability was affected.