Background:L-citrulline (L-Cit), a non-protein amino acid, has been implicated in several physiological functions including anti-inflammatory, anti-oxidative, and hypothermic roles, however, there is a paucity of information with regards to its potential in poultry production. Methods:This study was designed to investigate the effects of dietary L-Cit supplementation on the production performance, nitric oxide production, and antioxidant status of laying hens during summer period. Hy-Line Brown laying hens (n=288, 34 weeks old) were allotted to four treatment, 6 replicates of 12 chickens each. Dietary treatments of control (basal diets), 0.25％, 0.50％and 1.00％L-Cit supplementation were fed to chickens for eight (8) weeks. Production performance, free amino acid profiles, nitric oxide production, and antioxidant properties were measured. Blood samples were collected at the 4th and 8th weeks of the experiment. Results:Air temperature monitoring indicated an average daily minimum and maximum temperatures of 25.02 ℃and 31.01 ℃respectively. Dietary supplementation with L-Cit did not influence (P>0.05) the production performance, and rectal temperature of laying hens. Egg shape index was increased (P<0.05) with increasing levels of L-Cit. Serum-free content of arginine, citrulline, ornithine, tryptophan, histidine, GABA, and cystathionine were elevated, but taurine declined with L-Cit diets. Plasma nitric oxide (NOx) concentration was highest at 1％L-Cit. Likewise, nitric oxide synthase (NOS) activity for total NOS (tNOS) and inducible NOS (iNOS) were upregulated with increasing L-Cit levels, although, tNOS was not affected at the 4th week. Anti-oxidant enzymes including catalase and superoxide dismutase (SOD) were increased with L-Cit supplementation, however, SOD activity was unchanged at 4th week, while total anti-oxidant capacity increased at the 8th week. L-Cit supplementation attenuated the extent of lipid peroxidation, and also inhibited glutathione peroxidase activity. Conclusion:Dietary L-Cit supplementation modulated systemic arginine metabolism, nitric oxide synthesis, antioxidant defense system, and increased the egg shape index of laying hens during the summer season. 1％L-Cit supplementation proved most effective in potentiating these effects and may be adopted for feed formulation strategies.