Intestinal calcium absorption is markedly enhanced during pregnancy and lactation by prolactin (PRL) from the anterior pituitary. However, its cellular and molecular mechanisms pertaining to the intestinal calcium absorption are not completely understood. The present study aimed to demonstrate the transcriptome responses of intestinal epithelial cells to PRL in hyperprolactinemic animals, i.e., anterior pituitary (AP)-grafted rats and lactating rats, by using robotic spotting cDNA microarray and quantitative real-time PCR. In addition, the effects of PRL on proliferation and differentiation of IEC-6 crypt-like cells were also investigated. This study has been approved by the institutional animal care and use committees of Faculty of Science, Mahidol University. The results showed that several genes related to calcium absorption, e.g., transient receptor potential vanilloid calcium channel (TRPV)-5, TRPV6, calbindin-D9k, and claudin-3, were upregulated in the 4-week AP-grafted rat. Nevertheless, such the PRL action was observed only in the villous cells, but not in the IEC-6 crypt-like cells. PRL did not increase IEC-6 cell proliferation, suggesting that it did not contribute to the lactation-induced expansion of intestinal surface area. In early lactation, PRL was found to upregulate the expression of TRPV6 and plasma membrane Ca2+-ATPase-1b, which were apical and basolateral calcium transporters, respectively. Further microarray study revealed a number of changes in the expression of genes related to calcium, nutrient, and ion transport (such as TRPM7 and sodium-dependent phosphate transporter (NaPi)-IIb for magnesium and phosphate transport, respectively) in late lactation. In conclusion, PRL increased the intestinal calcium absorption by upregulating the expression of genes related to calcium transport, presumably to provide more calcium for milk production.