Diazinon (DZN) is an organophosphate insecticide has been used widely in agriculture to control pests. DZN has been found to induce reproductive dysfunctions in animals, particularly, avian species. This current study evaluated the lethal effects of acutely ingested DZN on the ovaries of laying hens. Twenty white leghorn laying hens were divided equally into two groups. Group one was kept as a control, and the second group was administrated orally with a single dose of DZN (6 mg/kg B.W) for twenty one days. At end of the experiment, serum and tissue samples (ovaries) were collected for hormonal and histopathological analysis. The results revealed that body weight was not affected significantly by DZN treatment. However, DZN treated group exhibited destructive changes in folliculogenesis, degeneration of ovarian follicles, and stromal hemorrhage in comparison with the control group. Additionally, significant changes of steroidal hormonal levels were observed with a significant increase in serum progesterone and decrease in estrogen in the treated group as compared to the control group. Therefore, the current reduction of serum estrogen in our study could be due to the destructive effects of DZN on ovarian tissues and/or its ability to alter the expression of P450 aromatase, the enzyme which is involved in estrogen production. In the contrary, serum progesterone levels increased significantly. This increase might be due to the activation of some steroidogenic enzymes such as 3β-hydroxy-steroid dehydrogenase (3β-HSD) by ovarian secondary follicles. In conclusion, this current study showed that acute oral administration of DZN induces destructive structural changes in ovary and changes in female sex steroid hormone levels in blood. This lethal effect of DZN may lead to the infertility in laying hens, thus economic lose and health impact.