Hyperosmolarity under physiological conditions resulting from lumen contents is a big challenge to the normal function of intestinal epithelium. Betaine is a potent osmolyte which is mostly studied in kidney. This study was aimed to gain insight into the osmoprotectant role of betaine in intestinal epithelium under hyperosmotic condition with intestinal porcine epithelial cells (IPEC-J2) and piglets. The result showed that betaine could attenuate the hyperosmolarity-induced reduction of cell viability after treating 12 h. Betaine ameliorates hyperosmotic stress in intestinal epithelium via decreasing reactive oxygen species (ROS) with the alleviation of cell shrinkage. Moreover, betaine prevented mitochondria from membrane collapse and alleviated mitochondrial-related apoptosis induced by hyperosmolarity in vivo and vitro. In addition, betaine ameliorated autophagy caused by hyperosmolarity by up-regulation LC3 II and down-regulation p62. In summary, betaine protects porcine small intestinal epithelium from hyperosmotic stress by alleviating cell shrinkage, ROS accumulation as well as apoptosis and autophagy.