Litchi thaumatin-like protein (LcTLP) has been identified as a predominant contributor to "Shanghuo". This study aimed to investigate the in vitro digestion and absorption characteristics of LcTLP and its regulatory effects on the intestinal barrier. By establishing a Caco-2 monolayer cell model, a RAW264.7 macrophage inflammation model, and a pseudo-germ-free mouse model, the transmembrane transport capacity and immune-activating effects of LcTLP were systematically analyzed, the regulatory effects of intestinal microbiota on LcTLP-induced intestinal morphological and metabolic alterations were also elucidated. The results indicated that in vitro digested LcTLP was absorbed at a rate of 24.53% in the Caco-2 model and was capable of penetrating the intestinal epithelial barrier effectively. Its absorbed products (50-200 ng·mL-1) were found to directly induce NO release from macrophages, with secretion levels elevated to 9.19-12.39 μmol·L-1 (1.80-2.43-fold of the control), indicating retained pro-inflammatory activity. Further, the pseudo-germ-free mouse model was used to observe the intestinal phenotype and metabolic response after LcTLP intervention under the background of microbiota depletion. The results showed that LcTLP treatment could induce an increase in fecal pH value and a decrease in the level of short-chain fatty acids (SCFAs), suggesting that LcTLP may affect the metabolic homeostasis of colonic microecology. In conclusion, LcTLP may jointly participate in the body''s stress response through trans-barrier immune activation and intestinal microenvironment perturbation.