β-Cell mass increases during pregnancy to accommodate for insulin resistance. This increase is mainly due to β-cell proliferation, a process that requires intact prolactin receptor (Prlr) signaling. Signaling molecules that are known to regulate β-cell proliferation include Jak2, Akt, the tumor suppressor menin, and cell cycle proteins. Whether these pathways are involved in prolactin-mediated β-cell proliferation is unknown. Using the heterozygous prolactin receptor-null (Prlr(+/-)) mice, we isolated pancreatic islets from both Prlr(+/+) and Prlr(+/-) mice on d 0 and 15 of pregnancy and examined the expression levels of these signaling molecules. In the wild-type mice (Prlr(+/+)), both phospho-Jak2 and phospho-Akt expression in pancreatic islets increased during pregnancy, which were attenuated in the pregnant Prlr(+/-) mice. During pregnancy, menin expression was reduced by 50 and 20% in the Prlr(+/+) and the Prlr(+/-) mice, respectively, and the pregnant Prlr(+/-) mice had higher islet p18 levels than the Prlr(+/+) mice. Interestingly, between d 0 and 15 of pregnancy, expression of cyclin inhibitory protein p21(cip) was increased in the Prlr(+/+) mice, but this increase was blunted in the Prlr(+/-) mice. Lastly, we did not find any difference in the expression levels of cyclins D1, D2, and inhibitory kinases between the pregnant Prlr(+/+) and Prlr(+/-) mice. Therefore, we conclude that during pregnancy, placental hormones act through the prolactin receptor to increase β-cell mass by up regulating β-cell proliferation by engaging Jak2, Akt, menin/p18, and p21. Future studies will determine the relative contribution of these molecules in maintaining normal glucose homeostasis during pregnancy.