The study was conducted the effects of NO on spleen lymphocyte apoptosis induced by selenium deficiency in chickens. Eighty one-day-old healthy chickens were randomly allocated into two groups (control group and selenium deficiency group) with four replicates and ten chickens in each replicate. Control group was fed a basal diet including 0.282 mg/kg Se, and selenium deficiency group was given a basal diet including 0.032 mg/kg Se. The samples were obtained from ten chickens at 30, 45, 60, 75 days of age in each group, and the activities of GSH-Px and NOS, the content of NO in spleen and serum, the content of selenium in spleen and whole blood, and the expression of iNOS mRNA and the lymphocyte apoptosis in spleen were detected. The results showed that, compared with control group, the content of selenium in spleen and whole blood and activity of GSH-Px in spleen and serum of the chickens in selenium deficiency group decreased while the content of NO, the activity of NOS and the expression of iNOS in spleen and serum increased, and the DNA of splenic lymphocyte was degraded and a ladder band appeared. Typical apoptosis was found in splenic lymphocyte of the chickens in selenium deficiency group by electron microscope, including cell shrinkage, nuclear chromatin margination and condensation, mitochondrial swelling and dissolution or disappearance of cristae, disorder and swelling of endoplasmic reticulum. It was concluded that selenium deficiency could induce the change of NO metabolism, and lead to apoptosis of splenic lymphocyte of chickens, and it further demonstrated NO played an important role in splenic lymphocyte apoptosis of chickens.