Objective: This study aimed to explore the effect of L-theanine on intestinal immunity of rats under resistant starch feeding. Methods: Different doses of L-theanine \[100, 300, 500 mg/（kg·d）\] was performed for 28 days in 4-week-old SD male rats by filling the stomach once daily under resistant starch feeding, in order to investigate the regulation of resistant starch combined with L-theanine on intestinal immunity. Results: The results were shown as follows the medium and high dose groups of L-theanine significantly increased the weight of rats, and improved the activities of ileum SOD, GSH-Px with reducing the content of MDA under resistant starch feeding; Pretreatment with L-theanine at different dosages improved the expressions of IL-4, IL-10, lowered the levels of IL-6, TNF-α, increased the level of IgA in ileum significantly. At the same time, L-theanine could heighten the contents of glutamate and glutamine in each small intestine, and improve the relative abundance of beneficial microorganisms such as Bifidobacterium, Prevotella_9, Blautia reducing the proportion of intestinal harmful microorganisms Helicobacter and Parasutterella （P＜0.05）. Consequently, the medium dose group of L-theanine \[300 mg/（kg·d）\] combined with resistant starch had the best effect. Conclusion: Under the resistant starch feeding, L-theanine can enhance intestinal immunity of rats by improving antioxidant capacity, balancing the levels of pro-inflammatory and anti-inflammatory factors and raisng the contents of glutamate and glutamine. In addition, it can also ameliorate the structure of intestinal bacteria to affect intestinal immunity.