ABSTRACT: The present study aimed to assess the impact of dietary valine levels on layer production performance, egg quality, immunity, and intestinal amino acid absorption of laying hens during the peak lay period. For this purpose, a total of 960 33-week-old Fengda No.1 laying hens were randomly divided into five experimental groups and fed with valine at the following different levels in a feeding trial that lasted 8 weeks: 0.59, 0.64, 0.69, 0.74, and 0.79%, respectively. Productive performances were recorded throughout the whole rearing cycle and the egg quality, serum indexes, and small intestine transporters expression were assessed at the end of the experiment after slaughter (41 weeks) on 12 hens per group. Statistical analysis was conducted by one-way ANOVA followed by LSD multiple comparison tests with SPSS 20.0 (SPSS, Chicago, IL, USA). The linear and quadratic effects were tested by SPSS 20.0. Egg mass, laying rate, broken egg rate, and feed conversion ratio were significantly improved with increasing dietary valine levels. However, the egg weight, eggshell thickness, albumen height, Haugh unit, and egg yolk color were significantly decreased with increasing dietary valine levels. Serum catalase (CAT), immunoglobulin A (IgA) and IgM levels, and malondialdehyde (MDA) levels were negative responses to valine-treated laying hens. Dietary supplemented valine enhanced the trypsin activity of duodenum chime and promoted the mRNA expression levels of ATB0,+, and LAT4 in the jejunum and corresponding serum free Ile, Lys, Phe, Val, and Tyr level. However, valine treatment significantly downregulated the mRNA expression levels of PePT1, B0AT1, LAT1, and SNAT2 in the small intestines and corresponding serum free Arg, His, Met, Thr, Ala, Asp, Glu, Gly, and Ser level. Our results suggest that 0.79% valine dietary supplementation can improve production performance by promoting amino acid nutrient uptake and utilization, and suggest a supplement of 0.79% valine to diet.