This study reports the effects of early-life lactoferrin (LF) intervention on the colonic microbiota, intestinal function and mucosal immunity in suckling piglets. A total of 60 Duroc x Landrace x Yorkshire suckling piglets from six sows were assigned to the control (CON) and LF groups in litters. The LF group piglets were fed 0.5 g/kg body weight of LF solution per day, and the CON group piglets were fed the same dose of physiological saline for a week. Six piglets from the two groups were randomly chosen and euthanised on days 8 and 21. The LF group piglets had higher ACE and Chao1 indices of colonic microbiota than the CON group piglets (P < 0.05). In addition, the LF group piglets had a higher abundance of Roseburia (P < 0.05) and a lower abundance of Escherichia-Shigella (P < 0.05) in the colonic digesta. The LF group piglets also had a higher concentration of butyrate (P < 0.05) in the colonic digesta. Moreover, the LF group piglets had a higher gene expression of occludin (P < 0.05) in the colonic mucosa. In addition, the gene expression of MUC4 was upregulated in the LF group piglets compared with that in the CON group on day 21 (P < 0.05), and the lower gene expression of TLR-4 was found in the LF group compared with the CON group on day 8 (P < 0.05). Furthermore, the concentration of IL-10 was increased in the LF group on day 8 (P < 0.05), while the LF group piglets had a higher concentration of sIgA and lower concentrations of IL-1 alpha and IL-1 beta (P < 0.05) in the colonic mucosa. These results suggest that early-life LF intervention can modulate the composition of colonic microbiota and improve the intestinal function in suckling piglets.