To our knowledge, there are no data in the literature regarding effects of excess dietary threonine on the laying hens during the summer climates. The goal of our study was to investigate the effects of threonine supplementation at levels higher than those recommended by the current NRC (1994) on production performance, the immune functions, serum free amino acids, blood indices, and gut functions. Babcock Brown layers (n=960),40wk of age, were allocated to5dietary treatments groups, each of which included6replicates of32hens. Each group received the same basal diet formulated with corn, peanut meal, and crystalline amino acids. Crystalline1-threonine (98.5%Thr, PT Cheil Jedang, Indonesia) was added to the basal diet at0(control),0.1,0.2,0.3, and0.4%for8wk. Dietary treatments were achieved by the addition of crystalline1-threonine at the expense of inert filler to give0.47[current NRC (1994) requirement],0.57,0.67,0.77, and0.87%threonine. Chemical analysis of the diets for threonine values are0.48,0.58,0.66,0.74, and0.85%, respectively. Egg production (EP), egg weight (EW), average daily feed intake (ADFI), and feed conversion ratio (FCR) were recorded in replicates. At48wk of age,18eggs from each treatment were randomly collected to assess egg quality parameters. At the end of the experiment (wk48), blood and tissue samples were collected from6hens (1bird/replicate). The results showed that although supplementing the diet with1-threonine did not affect ADFI, FCR, EW, or egg quality (P>0.05), the egg production response to supplemental1-threonine was quadratic, and it was maximized at0.2%supplemental1-threonine. No differences were observed for UA, LDH. ALP, Ca, and P concentrations among the treatments. Serum total protein concentration increased quadratically to supplemental threonine, and the response was maximized at0.2and 0.3%supplemental1-threonine. Serum free threonine increased quadratically as supplemental threonine increased, and the response was maximized between0.2and0.3%supplemental1-threonine. The addition of1-threonine at0.3%resulted in linearly increasing levels of IgG and total Ig (P<0.05) as compared with those of the control group. The numbers of goblet cells did not change due to L-threonine supplementation at0.2or0.3%as compared with control group. Also, L-threonine had no affect on the villus height and mucosal thickness at0.2or0.3%L-threonine. No differences were found among groups in the activity of ALP in jejunum or ileum. L-threonine supplementation at0.2%increased the level of serum SOD (P<0.05). Adding L-threonine had no affect on serum concentrations of MDA, GSH, GSH-Px, ACTH, GLU, and CHO. Expressions of jejunal and ileal mucin2mRNA were increased linearly by increasing1-threonine (P<0.01). The concentrations of sIgA antibody in the mucosa of the ileum increased linearly (P<0.01) at0.4%L-threonine and at0.3%1-threonine the concentrations of IgG antibody in the mucosa of the Jejunum tended to increase linearly (P=0.05). Although no differences attributable to treatment were found among groups in the activity of digestive enzymes (TRP and AMS) in the pancreas and mucosal homogenates of the jejunum and ileum, trypsin activity tended to increase quadratically (p=0.06) at0.2%L-threonine in the pancreas. Our findings suggest that L-threonine supplementation at0.2%maximized egg production quadratically and increased the level of serum SOD (P<0.05). Moreover, the addition of1-threonine at0.3%may have had a positive effect on the humoral immune response, whereas at0.4%the concentrations of slgA antibody in the mucosa of the ileum increased linearly (P<0.01). It was concluded that the control diet containing0.47%dietary threonine [current NRC (1994) requirement] recommendations are insufficient for modern commercial laying hens raised under high-temperature and high-humidity environmental climates. We suggest that dietary threonine requirement for laying hens ranged between0.66to0.74%levels under high-temperature and high-humidity environmental climates.