This study investigated the effects of the dietary threonine (Thr) levels on the performance, offspring traits, embryo amino acid transportation, and protein deposition in breeder hens of yellow-feathered chickens. In total, 720 breeder hens of Lingnan yellow-feathered chickens were randomly assigned to 1 of 6 dietary treatments, with 6 replicates per treatment (20 birds per replicate). The breeder hens were fed either basal diet (Thr = 0.38%) or basal diet supplemented with 0.12, 0.24, 0.36, 0.48, or 0.60% Thr from 197 to 266 D. There was a positive response in terms of the laying rate after adding different levels of Thr to the diet, but no significant effects on the average daily gain, average daily egg weight, feed conversion ratio, average broken eggs, and unqualified egg rate (P > 0.05). However, the eggshell strength and eggshell percentage decreased in a linear manner as the dietary Thr concentration increased (P = 0.05). Dietary supplementation with Thr had significant effects on the expression of mucin 2 (MUC2) in the uterus and zonula occludens protein 1 (ZO-1) in the duodenum of breeders (P < 0.05). In chick embryos at embryonic age 18 D, significant upregulation of poultry target of rapamycin (pTOR) occurred in the liver and breast muscle, as well as threonine dehydrogenase (TDH) in the thigh, and aminopeptidase (ANPEP) (P < 0.05) in the duodenum and ileum due to dietary Thr supplementation, but there were no effects on MUC2 expression in the duodenum and ileum (P > 0.05). The livability of the progeny broilers tended to increase with the dietary Thr concentration (quadratic, P = 0.08). Thus, dietary supplementation with Thr had positive effects on the laying production by breeder hens and offspring performance, and it also regulated the expression levels of genes related to amino acid transportation and protein deposition. The optimal dietary Thr concentration that maximized the laying rate in yellow-feathered chicken breeders aged 197 to 266 D was 0.68% according to quadratic regression analysis.