Prolactin (PRL) was previously demonstrated to rapidly enhance calcium absorption in rat duodenum and the intestinal-like Caco-2 monolayer. However, its mechanism was not completely understood. Herein, we investigated non-genomic effects of PRL on the transepithelial calcium transport and paracellular permselectivity in Caco-2 monolayer by Ussing chamber technique. PRL increased the transcellular and paracellular calcium fluxes, and paracellular calcium permeability within 60 min after exposure, but decreased the transepithelial resistance of the monolayer. The effects of PRL could not be inhibited by RNA polymerase II inhibitor (5,6-dichloro-1--D-ribobenzimidazole), confirming that PRL actions were non-genomic. Exposure to protein kinase C (PKC) or RhoA-associated coiled-coil forming kinase (ROCK) inhibitors (GF109203X and Y27632, respectively) abolished the stimulatory effect of PRL on the transcellular calcium transport, whereas ROCK inhibitor, but not PKC inhibitor diminished PRL effect on the paracellular calcium transport. Knockdown of the long isoform of PRL receptor (PRLR-L) also prevented the enhancement of calcium transport by PRL. In addition, PRL markedly increased paracellular sodium permeability and permeability ratio of sodium to chloride, which are indicators of the paracellular charge-selective property, and are known to be associated with the enhanced paracellular calcium transport. The permeability of other cations in the alkali metal series was also increased by PRL, and such increases were abolished by ROCK inhibitor. It could be concluded that PRL stimulated the transepithelial calcium transport through PRLR-L, and increased the paracellular permeability to cations in Caco-2 monolayer. These non-genomic actions of PRL were mediated by the PKC and ROCK signaling pathways.