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Introduction: Nigella sativa (N. sativa), one of the most commonly used medicinal herbs with antioxidant properties, increases blood insulin levels and lowers fasting blood sugar. Nuclear Erythroid Factor-Related Factor 2 (Nrf2) and Fibroblast Growth Factor 21 (FGF21) are two antioxidant factors that are increased by oxidative stress and hyperglycemia. The present study investigated how hydroalcoholic extract of N. sativa seed (HENS) increases blood insulin levels, taking into account changes in antioxidant factors and expression of insulin transcription factors. Materials and methods: Two groups of male diabetic wistar rats were treated orally with HESN at doses of 200 and 400 mg/kg-body weight for one month. Fasting blood sugar (FBS) and insulin were measured using standard kits by photometric and ELISA methods, respectively. The expression levels of the Nrf2, FGF21 and β-Klotho genes as well as the insulin gene-stimulating transcription factors of MafA and PDX-1 were evaluated using real-time PCR. Oxidative stress was assessed by assessing serum total oxidation status (TOS), malondialdehyde (MDA), and total antioxidant capacity (TAC). Results: HSEN showed a significant reducing effect on FBS and oxidative biomarkers and an increasing effect on serum insulin levels in treated diabetic rats compared to untreated diabetics (P < 0.05). The elevated levels of NRF2 and FGF21 in the liver and pancreas of the diabetic control group were significantly reduced after treatment with both HESN doses (P < 0.05). Following the ameliorative effects of HENS on pancreatic tissue and the reduction of oxidative stress, the expression level of MafA and PDX1 genes approached the level of these factors in healthy rats (P < 0.05). Conclusion: This study showed the therapeutic effects of HENS on diabetic pancreas by reducing oxidative stress and tissue damage, modifying the expression levels of PDX-1 and MafA genes, and regulating insulin secretion and blood glucose levels. [ABSTRACT FROM AUTHOR]