Prolactin (PRL) is reported to stimulate calcium absorption in the rat's small intestine. However, little is known regarding its effects on the cecum, a part of the large intestine with the highest rate of intestinal calcium transport. We demonstrated herein by quantitative real-time polymerase chain reaction and Western blot analysis that the cecum could be a target organ of PRL since cecal epithelial cells strongly expressed PRL receptors. In Ussing chamber experiments, PRL enhanced the transcellular cecal calcium absorption in a biphasic dose-response manner. PRL also increased the paracellular calcium permeability and passive calcium transport in the cecum, which could be explained by the PRL-induced decrease in transepithelial resistance and increase in cation selectivity of the cecal epithelium. PRL actions in the cecum were abolished by inhibitors of phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK), but not inhibitors of gene transcription and protein biosynthesis. In conclusion, PRL directly enhanced the transcellular and paracellular calcium transport in the rat cecum through the nongenomic signaling pathways involving PI3K, PKC, and ROCK.