Objective To investigate the effects of lactoferrin (LF) deficiency during lactation and LF supplementation from different sources on intestinal development of newborn mice.Methods Female LF knockout mice were used to produce LF deficiency milk for newborn mice intake during lactation, and 100 mg/kg bovine Lactoferrin (bLF), recombinant human Lactoferrin (rhLF) and bovine serum albumin (BSA) were artificially fed every day from the 3rd day to the 21st day. Samples were taken at the age of 21 days to determine the small intestine development index of mice in each group.Results In this experimental cycle, rhLF supplementation significantly increased the villus length/crypt depth of ileum (P<0.05), up-regulated the expression of Occludin and ZO-1 in ileum (P<0.05) and increased the ratio of maltase activity/lactase activity in duodenum (P<0.05), jejunum (P<0.05) and ileum (P<0.05) of mice. In addition, the supplementation of bLF only significantly increased the ratio of maltase activity/lactase activity in ileum (P<0.05), indicating that the supplementation of LF during lactation can promote the intestinal development of newborn mice.Conclusion For newborn mouse without LF intake during lactation, the supplement of LF during lactation can enhance the intestinal digestion and absorption of nutrients, promote the development and maturity of the intestinal tract, and enhance the intestinal barrier function. Moreover, rhLF plays a more effective role than bLF.