The pituitary hormone prolactin (PRL) is a multifunctional polypeptide which act as a key component of the neuroendocrine-immune loop and as a local regulator of the macrophage response. The involvement of PRL in regulating monocyte/macrophage functions is suggested by the presence of PRL receptors in these cells. Recently, we reported that physiological concentrations of native PRL were able to induce the expression of the pro-inflammatory cytokines IL-1β and TNFα, and the production of reactive oxygen species (ROS) in head kidney leukocytes and macrophages from the teleost fish gilthead seabream (Sparus aurata L.). In this study, we show that the NADPH oxidase subunit p47phox becomes phosphorylated in leukocytes stimulated with PRL, an effect that is blocked when neutralizing polyclonal antibodies to PRL are added. Additionally, the pharmacological inhibition of either protein kinase C (PKC) with calphostin C or the Jak/Stat signaling pathway with AG490 impaired PKC activation, p47phox phosphorylation and ROS production in seabream leukocytes activated with PRL. Taken together, our results demonstrate for the first time the need for PKC in regulating the PRL-mediated phosphorylation of p47phox, the activation of NADPH oxidase and the production of ROS by macrophages in vertebrates.