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Lactoferrin is a natural compound in the milk of mammals and was shown to influence the intestinal micro-flora and the immune system in mice, calves, dogs and man. The present study was carried out to investigate the effect of orally administered bovine lactoferrin (0, 30, 60 and 120 mg/kg DM feed) on the intestinal morphology and lymphocyte colonization in 36 motherless raised puppies. Endoscopic biopsies from duodenum and colon, taken in week 14, were scored histologically after staining with haematoxylin and eosin (H&E) and lymphocytes (CD3+, CD4+, CD8+) and plasma cells (IgA+, IgG+, IgM+) were enumerated after immunohistochemical staining by computer-aided quantification. Histological scoring revealed no significant differences amongst the groups. IgG+ plasma cells were reduced (p < 0.05) in the lamina propria of the colon of the 30 and the 60 mg group. The number of CD8+ lymphocytes was higher (p < 0.05) in the epithelium of the colon of the lactoferrin groups. In conclusion, this study indicated only minimal effects of bovine lactoferrin on the population of selected immune cells in the gut mucosa of puppies. More investigations are needed to describe the impact of lactoferrin on the digestive physiology of puppies.