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Aim: We aimed to examine the alterations of the insulin signaling pathway, autophagy, nitrative stress and the effect of vitamin D supplementation in the liver and ovaries of vitamin D deficient hyperandrogenic rats. Methods: Female Wistar rats received eight weeks of transdermal testosterone treatment and lived on a low vitamin D diet (D&ndash, T+). Vitamin D supplementation was achieved by oral administration of vitamin D3 (D+T+). Sham-treated (D+T&ndash, ) and vitamin D deficient animals (D&ndash, T&ndash, ) served as controls. (N = 10&ndash, 12 per group). Results: D&ndash, T+ animals showed decreased LC3 II levels in the liver and increased p-Akt/Akt and p-eNOS/eNOS ratios with decreased insulin receptor staining in the ovaries. Vitamin D supplementation prevented the increase of Akt phosphorylation in the ovaries. Vitamin D deficiency itself also led to decreased LC3 II levels in the liver and decreased insulin receptor staining in the ovaries. D&ndash, T+ group showed no increase in nitrotyrosine staining, however, the ovaries of D&ndash, rats and the liver of D+T+ animals showed increased staining intensity. Conclusion: Vitamin D deficiency itself might lead to disrupted ovarian maturation and autophagy malfunction in the liver. Preventing Akt phosphorylation may contribute to the beneficial effect of vitamin D treatment on ovarian function in hyperandrogenism.