This study evaluated the effects of leptin on primordial follicle survival and activation after in vitro culture of ovine ovarian tissue and if leptin acts through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. Ovarian fragments were fixed for histology (fresh control) or cultured for 7 days in control medium (α-MEM+) alone or supplemented with leptin (1, 5, 10, 25 or 50 ng/ml). Follicle morphology, activation and apoptosis were analyzed. Next, the fragments were cultured in the medium that showed the best results in the absence or the presence of the PI3K inhibitor (LY294002), and immunohistostaining of p-Akt protein was assessed. After culture, the percentage of normal follicles decreased (P < 0.05) in all treatments compared with the fresh control. Moreover, control medium and 1 ng/ml leptin had similar (P > 0.05) percentages of normal follicles, which were significantly higher than those in other treatments. However, culture with 1 ng/ml leptin maintained apoptosis similarly (P > 0.05) to that of the fresh control and lower (P < 0.05) than that in α-MEM+. Leptin did not influence follicle activation (P > 0.05) compared with the control medium (α-MEM+). Culture in 1 ng/ml leptin with LY294002 decreased the normal follicles and increased apoptosis, inhibited follicle activation (P < 0.05), and reduced p-Akt immunostaining, compared with the medium containing 1 ng/ml leptin without PI3K inhibitor. In conclusion, leptin at 1 ng/ml reduces apoptosis and promotes the activation of primordial follicles compared with the fresh control after in vitro culture of ovine ovarian tissue possibly through the PI3K/Akt pathway.