The present study evaluated the effects of mercury chloride (HgCl2) on follicular atresia rate, sex hormone secretion, and ovarian oxidative stress in laying hens. Antioxidant enzyme genes and the nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signal pathway were further studied to uncover the molecular mechanism. A total of 768 40-week-old Hy-Line Brown laying hens were randomly allocated to four treatments with eight pens per treatment and 24 hens of each pen. The birds were fed with four experimental diets containing graded levels of mercury (Hg) at 0.280, 3.325, 9.415, and 27.240 mg/kg, respectively. Results revealed that a positive relationship occurred between the accumulation of Hg in ovary and follicular atresia rate. Progesterone (P4) level significantly decreased in all Hg-treatment groups (P < 0.05), and follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were the lowest in the 27.240-mg/kg Hg group. Besides, the activities of catalase (CAT), superoxidative dismutase (SOD), glutathione reductase (GR), and glutathione (GSH) content were significantly decreased in all Hg-treatment groups (P < 0.05). Glutathione peroxidase (GSH-Px) activity significantly decreased, while malondialdehyde (MDA) content sharply increased in the 27.240-mg/kg Hg group (P < 0.05). In addition, there were positive relationships between antioxidant enzyme activities and antioxidant gene expressions or between antioxidant gene expressions and Nrf2 mRNA expression, while negative correlations occurred between Nrf2 and Keap1 at transcription and protein levels. It could be concluded that Hg induced ovarian function disorders and ovarian oxidative stress by means of impairing the Nrf2-Keap1 signal pathway in laying hens.