During pregnancy and lactation, the enhanced intestinal Ca(2+) absorption serves to provide Ca(2+) for fetal development and lactogenesis; however, the responsible hormone and its mechanisms remain elusive. We elucidated herein that prolactin (PRL) markedly stimulated the transcellular and paracellular Ca(2+) transport in the duodenum of pregnant and lactating rats as well as in Caco-2 monolayer in a two-step manner. Specifically, a long-term exposure to PRL in pregnancy and lactation induced an adaptation in duodenal cells at genomic levels by upregulating the expression of genes related to transcellular transport, e.g., TRPV5/6 and calbindin-D(9k), and the paracellular transport, e.g., claudin-3, thereby raising Ca(2+) absorption rate to a new "baseline" (Step 1). During suckling, PRL surge further increased Ca(2+) absorption to a higher level (Step 2) in a nongenomic manner to match Ca(2+) loss in milk. PRL-enhanced apical Ca(2+) uptake was responsible for the increased transcellular transport, whereas PRL-enhanced paracellular transport required claudin-15, which regulated epithelial cation selectivity and paracellular Ca(2+) movement. Such nongenomic PRL actions were mediated by phosphoinositide 3-kinase, protein kinase C, and RhoA-associated coiled-coil-forming kinase pathways. In conclusion, two-step stimulation of intestinal Ca(2+) absorption resulted from long-term PRL exposure, which upregulated Ca(2+) transporter genes to elevate the transport baseline, and the suckling-induced transient PRL surge, which further increased Ca(2+) transport to the maximal capacity. The present findings also suggested that Ca(2+) supplementation at 15-30 min prior to breastfeeding may best benefit the lactating mother, since more Ca(2+) could be absorbed as a result of the suckling-induced PRL surge.