The role of sex steroids in the modulation of fish immune responses has received little attention. Previous studies have demonstrated that 17β-estradiol (E(2)) is able to alter the response of gilthead seabream leukocytes to infectious agents. We have used suppression subtractive hybridization to identify genes upregulated by E(2) (50 ng/ml) in macrophage cultures from gilthead seabream. We isolated 393 up-regulated cDNA fragments that led to the identification of 162 candidate estrogen-responsive genes. Functional analyses revealed the presence of several enriched immune processes and molecular pathways. The E(2) up-regulation of some immune-relevant genes was further confirmed by real time RT-PCR. Bioinformatics analysis revealed the ability of E(2) to orchestrate profound alterations in the macrophage expression profile, especially immune-related processes and pathways. This is the first report on E(2)-dependent modifications of fish macrophage transcriptome and lends weight to a suggested role for estrogen in the immune system, the possible significance of which is discussed.