Purpose Threonine (Thr) can be involved in the synthesis of immunoglobulins, which play the role of immune regulation, Thr also has to improve intestinal morphology, adjust the sticky protein synthesis, maintain the intestinal barrier function, etc. The experiment aimed to investigate the effects of diets supplemented with different levels of Thr on growth performance and intestinal health of rabbits under lipopolysaccharide (LPS) stress conditions. Methods A total of 180 healthy 35-day-old weaned New Zealand White rabbits were randomly assigned in a 2 x 3 factorial design to receive an intraperitoneal injection of 100 mu g/kg BW LPS or saline and three diets with different levels of digestible threonine (0.43%, 0.54%, and 0.64%). Results The LPS challenge resulted in a reduction in body weight in rabbits at day 22, as well as a decrease in the serum d-lactic acid (D-LA) content and the number of goblet cells (GCs) in the jejunum. Additionally, the duodenum JAM2 and JAM3 were down-regulated. The expression of OCLN, ZO-1, and IL-2 in the jejunum, and CLDN, nuclear factor-kappa B (NF-kappa B) and ZO-1 mRNA in the ileum were also down-regulated. Furthermore, the duodenum TLR4 and IL-1 beta mRNA expression, while the jejunum exhibited an elevation in CLDN, TNF-alpha, and ileum TNF-alpha mRNA expression (P < 0.05). In the context of LPS challenge condition, dietary Thr addition was found to down-regulate the duodenum ZO-1 and jejunum CLDN mRNA expression of rabbits (P < 0.05). This was accompanied by an increase in ileum sIgA content and GCs number (P < 0.05). Additionally, dietary Thr addition resulted in a downregulation of duodenum TLR4, MyD88, NF-kappa B, TNF-alpha and IL-1 beta, jejunum MyD88, and IL-1 beta mRNA expression, as well as an up-regulation of ileum IL-10 mRNA expression in rabbits (P < 0.05). Conclusion In conclusion, the LPS challenge can result in intestinal inflammation and damage the integrity of the intestinal barrier in rabbits. Nevertheless, dietary Thr supplementation can alleviate the intestinal inflammatory response in rabbits challenged with LPS.