ANG4 (angiogenin4), a novel antimicrobial peptide with antimicrobial activity and stimulating angiogenesis function mainly expressed in intestinal tissues of mammals, plays a key role in immune response of animal against pathogens and angiogenesis. Regarding to the multiple functions, ANG4 has a very broad prospect in the prevention and treatment of intestinal diseases of piglets. To determine the expression pattern of ANG4 in newborn piglet’s intestine, the mRNA and protein levels of ANG4 as well as the cellular localization were analyzed by real-time PCR, western blot, and immunohistochemistry respectively. The results showed that the change tendency of ANG4 in different parts of the piglet’s intestine was approximately the same. In the same tissue, the expression level of ANG4 gradually increased with the increase of the age of the week, and it increased significantly from the third week to the fifth week. In addition, during the same period, the expression of ANG4 in the ileum and colon was higher, followed by the jejunum, and the expression was lowest in the duodenum. The expression of ANG4 protein is mainly concentrated in the small intestine villi and crypt. The main expression cells are enterocytes and Paneth cells.