Because of rare glucagon-like peptide-2 (GLP-2) receptor (+) cells within the gut mucosa, the molecular mechanisms transducing the diverse actions of GLP-2 remain largely obscure. This research identified the naturally occurring intestinal cell lines that endogenously express GLP-2R and determined the molecular mechanisms of the protective effects of GLP-2-mediated tight junctions (TJ) in GLP-2R (+) cell line. (i) Immunohistochemistry results showed that GLP-2R is localised to the epithelia, laminae propriae and muscle layers of the small and large bowels of newborn piglets. (ii) GLP-2R expression was apparent in the cytoplasm of endocrine cells in IPEC-J2 cell lines. (iii) The protein expressions of ZO-1, claudin-1, occludin, p-PI3 K, p-Akt, p-mTOR and p-p70S6K significantly (p < 0.05) increased in GLP-2-treated IPEC-J2 cells, and all of them significantly (p < 0.05) decreased when LY-294002 or rapamycin was added. GLP-2 improves intestinal TJ expression of GLP-2R (+) cells through the PI3 k/Akt/mTOR/p70S6K signalling pathway.