Prolactin (PRL) was originally identified by its ability to stimulate mammary development and lactation, and its essential roles other than lactation have recently been implicated in female reproduction. However, little is known about PRL-mediated events in pregnancy. The tryptophan catabolism enzyme indoleamine 2,3-dioxygenase (IDO) is interferon-gamma (IFN-gamma)-inducible and has recently become a focus for maternal-fetal tolerance for successful pregnancy. Based on recognition that PRL is one of the up-regulated hormones in pregnancy, in a previous study we have shown that PRL induces IDO expression in monocytes in cooperation with a suboptimal concentration of IFN-gamma. Here, we demonstrate that PRL sensitizes monocytes to induce IDO expression in response to low doses of IFN-gamma without affecting the typical IFN-gamma signaling events, such as STAT1 phosphorylation and IRF-1 induction. In addition, IDO induction in these cell cultures was observed only after 24 h pre-exposure to PRL. These results indicate a priming effect of PRL on monocytes that occurs before IFN-gamma signaling and increases their sensitivity to IFN-gamma for IDO induction, rather than a synergistic effect of PRL and IFN-gamma on IDO induction. These results offer new insights into the roles of PRL in female reproduction, as well as provide a better understanding as to how IDO expression is regulated and achieved in pregnancy.