Previous investigations suggested that prolactin (PRL) stimulated the intestinal calcium absorption through phosphoinositide 3-kinase (PI3K), protein kinase C (PKC) and RhoA-associated coiled-coil forming kinase (ROCK) signaling pathways. However, little was known regarding its detailed mechanisms for the stimulation of transcellular and voltage-dependent paracellular calcium transport. By using Ussing chamber technique, we found that the PRL-induced increase in the transcellular calcium flux and decrease in transepithelial resistance of intestinal-like Caco-2 monolayer were not abolished by inhibitors of gene transcription and protein biosynthesis. The PRL-stimulated transcellular calcium transport was completely inhibited by the L-type calcium channel blockers (nifedipine and verapamil) and plasma membrane Ca²⁺-ATPase (PMCA) inhibitor (trifluoperazine) as well as siRNA targeting L-type calcium channel Ca_v1.3, but not TRPV6 or calbindin-D_9k. As demonstrated by ⁴⁵Ca uptake study, PI3K and PKC, but not ROCK were essential for the PRL-enhanced apical calcium entry. In addition, PRL was unable to enhance the transcellular calcium transport after PKC knockdown or exposure to inhibitors of PKC, but not of PKC, PKC, PKC, PKC_µ or protein kinase A. Voltage-clamping experiments further showed that PRL markedly stimulated the voltage-dependent calcium transport, and removed the paracellular rectification. Such PRL effects on paracellular transport were completely abolished by inhibitors of PI3K (LY294002) and ROCK (Y27632). It could be concluded that the PRL-stimulated transcellular calcium transport in Caco-2 monolayer was mediated by Ca_v1.3 and PMCA, presumably through PI3K and PKC pathways, while the enhanced voltage-dependent calcium transport occurred through PI3K and ROCK pathways.