The intestine is a key organ for the absorption of amino acids. L-theanine (LTA) is a structural analog of glutamine and a characteristic non-protein amino acid found in tea (Camellia sinensis) that regulates lipid and protein metabolism. The present study explored the role of LTA in intestinal amino acid absorption, protein synthesis, and its mechanisms. Overall, our findings suggest that LTA supplementation not only affects serum alkaline phosphatase (AKP), total protein (TP), and urea nitrogen (BUN) levels, but it also upregulates the mRNA and protein expression of amino acid transporters (EAAT3, EAAT1, 4F2hc, y+LAT1, CAT1, ASCT2, and B0AT1), and activates the mTOR signaling pathway. The downstream S6 and S6K1 proteins are regulated, and the expression of amino acid transporters is regulated. These findings suggest that LTA increases intestinal AA absorption, promotes protein metabolism, and increases nitrogen utilization by upregulating AAT expression, activating the mTOR signaling pathway, and phosphorylating the mTOR downstream proteins S6 and S6K1.