In lactation, the intestinal absorption of nutrients and minerals, especially calcium, is markedly enhanced to supply precursors for milk production. Little is known regarding the mechanism of this lactation-induced intestinal hyperabsorption. However, it has been postulated to result from villous hypertrophy with enlarged absorptive area and the upregulation of the cation-selective tight junction protein claudin-15, which could form calcium-permeable paracellular pores, thereby enhancing the paracellular calcium absorption. Here, we demonstrated in the duodenum of 21-day lactating rats that there were increases in the villous height, villous width and crypt depth, which together led to expansion of absorptive surface area. Quantitative real-time PCR further showed that the mRNA levels of claudin-10 and -15 were increased in the duodenal mucosal cells of lactating rats as compared to age-matched unmated control rats. However, immunohistochemical analysis revealed the lactation-induced upregulation of claudin-15, but not claudin-10 protein expression in the duodenal villous cells. The present results, therefore, corroborated the previous hypothesis that lactation induced intestinal absorption of calcium and perhaps other cation minerals, in part, by increasing villous absorptive area and claudin-15 protein expression.