Lactoferrin is a protein with multifunctional bioactivities. However, limited studies have reported the digestion behavior of lactoferrin in mice. In the present study, the metabolic fate of lactoferrin in the gastrointestinal tract (GIT) of mice was investigated. The mice were orally administered 4 mg of lactoferrin. The contents in the stomach, duodenum, jejunum and ileum were collected at 0.5 h, 1 h, 2 h and 3 h after administration. The peptides in the contents of the stomach, duodenum, jejunum and ileum were identified by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) coupled with capillary electrophoresis (CE). At 0.5 h after administration, 350, 47, 118 and 26 novel peptides were identified in the contents of the stomach, duodenum, jejunum and ileum, respectively. At 1 h after administration, 175, 23, 3 and 8 novel peptides were identified in the contents of the stomach, duodenum, jejunum and ileum, respectively. At 2 h and 3 h after administration, 96 and 2 novel peptides were identified in the contents of the stomach, respectively. Among these peptides, 10 peptides were generated from gastrointestinal digestions and could play physiological roles when they are absorbed through the intestine in their original form. This work provided some theoretical basis for clarifying the bioactive mechanisms of lactoferrin.