During pregnancy and lactation, the enhanced intestinal Ca²⁺ absorption serves to provide Ca²⁺ 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 Ca²⁺ transport in the duodenum of pregnant and lactating rats as well as in Caco-2 monolayer in a 2-step manner. Specifically, a long-term exposure to PRL in pregnancy and lactation induced an adaptation in duodenal cells at genomic level 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 Ca²⁺ absorption rate to a new "baseline" (Step 1). During suckling, PRL surge further increased Ca²⁺ absorption to a higher level (Step 2) in a non-genomic manner to match Ca²⁺ loss in milk. PRL-enhanced apical Ca²⁺ uptake was responsible for the increased transcellular transport, whereas PRL-enhanced paracellular transport required claudin-15, which regulated epithelial cation selectivity and paracellular Ca²⁺ movement. Such non-genomic PRL actions were mediated by phosphoinositide 3-kinase, protein kinase C, and RhoA-associated coiled-coil forming kinase pathways. In conclusion, 2-step stimulation of intestinal Ca²⁺ absorption resulted from long-term PRL exposure which upregulated Ca²⁺ transporter genes to elevate the transport baseline, and the suckling-induced transient PRL surge which further increased Ca²⁺ transport to the maximal capacity. The present findings also suggested that Ca²⁺ supplementation at 15-30 min prior to breastfeeding may best benefit the lactating mother since more Ca²⁺ could be absorbed as a result of the suckling-induced PRL surge.