At weaning, mammals switch from milk to complex adult food, and change from a lactose-rich to a lactose-free diet. At the same time, the small intestine matures resulting in changes in lactase expression and the onset of sucrase. The aim of this study was to analyze the effect of premature and specific depletion of lactose on maturation of the small intestine and on lactase expression in suckling mice. For this purpose, from postnatal days 10 to 16, suckling mice were fed by transgenic alpha-lactalbumin-deficient females that produce lactose-free milk. Pups fed with lactose-free milk had a lower body weight than controls fed by wildtype females. They also displayed hypotrophy of intestinal muscle layers, but no obvious alterations in the morphology of the intestinal epithelium. The level of lactase activity as well as the longitudinal distribution of corresponding mRNA were unchanged compared to suckling animals nourished with normal lactose-containing milk. Finally, there was no premature onset of sucrase expression. We conclude that feeding suckling mice for six days with lactose-free milk does not provoke any premature maturation of the small intestine. Thus, decreasing lactose intake is not a major cause for the modifications of lactase expression which occur at weaning.