A higher concentration of melatonin (MEL) in the intestine — even more than that in the plasma and pineal gland — implies its putative important role in the gastrointestinal structural or functional regulation. However, little evidence has shown that MEL can regulate the physiological functions of the intestinal mucosa. In this study, fertilized chicken eggs were treated with MEL (0.1 to 10 μg/d) from embryonic d 12 (E12) to post-hatching d 6 (D6), and the small intestine samples were collected at D6 to determine the changes in mucosal construction and function. Results of HE staining showed that the enterocyte number was not changed after MEL treatment. Alcian blue - periodic acid Schiff reaction (AB-PAS) staining and qRT-PCR showed that the goblet cells populations and mucins gene (MUC2) expression in the small intestine were significantly increased after MEL treatment. Meanwhile, 5-ethynyl-2′-deoxyuridine staining showed that both the proliferation and migration rates of the small intestine mucosal epithelium were promoted by MEL treatment. Importantly, MEL significantly increased the activities of the digestive enzymes (maltase, sucrose, and lactase) and expression of the nutrient transporter genes such as GLUT5, BOAT, and EAAT3 mRNAs in the duodenum or jejunum. Meanwhile, the expression of Notch receptors (Notch1 and Notch2) and their ligands (Dll1 and Dll4) were remarkably decreased after MEL treatment. In conclusion, MEL treatment increased the goblet cell populations, MUC2 expression, epithelium migration, and digestive and absorptive function of the chicken small intestine involving repressed Notch signaling.