Prolactin (PRL) has been shown to stimulate intestinal calcium absorption but the mechanism was still unknown. This study aimed to investigate the mechanism and signaling pathway by which PRL enhanced the calcium transport in the rat duodenum and Caco-2 monolayer. Both epithelia strongly expressed mRNAs and proteins of PRL receptors. Ussing chamber technique showed that the duodenal active calcium fluxes were increased by PRL in a dose-response manner with the maximal effective dose of 800 ng/mL. This response diminished after exposure to LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Caco-2 monolayer gave similar response to PRL with the maximal effective dose of 600 ng/mL. By nullifying the transepithelial potential difference, we showed that the voltage-dependent paracellular calcium transport did not contribute to the PRL-enhanced flux in Caco-2 monolayer. In contrast, the calcium gradient-dependent paracellular transport and calcium permeability were increased by PRL. Effects of PRL on Caco-2 monolayer were abolished by PI3K inhibitors (LY294002 and wortmannin), but not by inhibitors of MEK (U0126) or JAK2 (AG490). To investigate whether the PRL-enhanced paracellular transport was linked to changes in the epithelial charge selectivity, the permeability ratio of sodium and chloride (P_Na/P_Cl) was determined. We found that PRL elevated the P_Na/P_Cl in both epithelia, and the effects were blocked by PI3K inhibitors. In conclusion, PRL directly and rapidly stimulated the active and passive calcium transport in the rat duodenum and Caco-2 monolayer via the non-genomic PI3K signaling pathway. This PRL-enhanced paracellular calcium transport could have resulted from altered charge selectivity.