Prolactin (PRL), a peptide hormone produced by the pituitary gland, was shown to play an important role in the modulation of the immune system of lower and higher vertebrates. To further investigate the effects of PRL on the activation of professional phagocytes of bony fish, we stimulated head kidney leukocytes and purified macrophages from the gilthead seabream (Sparus aurata L.) with various physiological concentrations of native salmon PRL for 2 and 16 h and analyzed the respiratory burst activity and proinflammatory cytokine expression profile. The results showed that PRL was able to induce the production of reactive oxygen species and the expression of IL-1 beta and TNF-alpha in a similar way to two major pathogen-associated molecular patterns: polyinosinic-polycytidylic acid and genomic DNA from the bacterium Vibrio anguillarum. Interestingly, when the leukocytes were stimulated with suboptimal concentrations of PRL in the presence of bacterial DNA, the expression of IL-1 beta was synergistically induced. More importantly, all PRL activities were blocked by neutralizing Abs to PRL, as well as by pharmacological inhibitors of the Jak/Stat and NF-kappa B signaling pathways. In addition, EMSA and HPLC/mass spectrometry further confirmed that Stat and NF-kappa B were involved in the activation of seabream leukocytes by PRL. Collectively, our data identified PRL as a key regulator of the activation of fish professional phagocytes and demonstrated a cross-talk between TLR/NF-kappa B and PRLR/Jak/Stat signaling pathways. In addition, to the best of our knowledge, this is the first report showing that PRL modulates the activation of phagocyte NADPH oxidase through the Jak/Stat pathway in vertebrates. The Journal of Immunology, 2010, 185: 3873-3883.