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Background: Gestational diabetes mellitus has a long-term effect on pregnant women. Walnut (Juglans regia L.) oil-derived polyunsaturated fatty acid (PUFA) possesses multifarious pharmacological activities. This study investigated the beneficial effects of walnut oil-derived PUFA on glucose metabolism, pregnancy outcomes, oxidative stress, and lipid metabolism in gestational diabetes mellitus. Methods: The GDM rat model was generated by intraperitoneal injection of streptozotocin (40 mg/kg) on gestational day (GD) 6, GD7 and GD8. The differences between groups were estimated using one-way ANOVA followed by the Tukey's multiple comparison test for post-hoc analysis. Results: The results indicated that PUFA could mitigate GDM in pregnant diabetic rats, as embodied by the decrease of fasting blood glucose and the increase of plasma insulin and hepatic glycogen levels. Also, PUFA could suppress oxidative stress in pregnant diabetic rats, as reflected by the decrease of malondialdehyde content, an increase of superoxide dismutase, catalase and gutathione peroxidase activities. PUFA could also mitigate the abnormal changes of lipid profiles in plasma and hepatic tissue. Moreover, the relative mRNA expression of sterol regulatory element-binding transcription factor-1, stearoyl-CoA desaturase-1, fatty acid synthase, and acetyl-coenzyme A carboxylase, was suppressed by PUFA in pregnant diabetic rats. Conclusions: These results suggested that PUFA supplementation during pregnancy is beneficial in preventing diabetic complications in pregnant rats. [ABSTRACT FROM AUTHOR]