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17α-ethinylestradiol (EE2) exerts endocrine disrupting effect and immunotoxic effect on marine animals, including modulation of hepcidin expression. The antimicrobial peptide hepcidin displays a crucial role in innate immunity in fish against invading pathogens. It is known that the transcription of hepcidin in mammals is individually regulated by many stimuli, including inflammation, iron overload, anemia or hypoxia, through several distinct molecular pathways. The canonical mechanism for endocrine disrupting effects is mediated by an estrogen receptor (ER) and estrogen responsive element (ERE), whereas the underlying mechanism for immunotoxic effect is still unclear. In this study, a hepcidin from Oryzias melastigma (OM-hep1) was found to be down-regulated upon EE2 exposure and was associated with ERα. Unlike the revealed signaling pathways for hepcidin regulation in mammals, it was revealed by promoter activity analysis that the OM-hep1 transcription was not associated with canonical immune-associated and hormone-associated regulatory elements, known as the nuclear factor κB (NF-κB), signal transducer and activator of transcription 3 (STAT3), ERE and estrogen-related receptor responsive element (ERRE). Further analysis through a series of base mutations revealed a short fragment from −315 to −289 bp on the OM-hep1 promoter with high activity. This fragment was composed of a putative ERE-like element (23 bases) plus an adjacent down-streamed four bases motif GTGT. Replacement of either of the core bases (GGTCA) of ERE-like or GTGT motif showed non-activity and non-response to EE2 exposure, thus a new hepcidin-associated element named as HepERE was revealed. Evidences from electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) assay demonstrated that the EE2-mediated down-regulation of OM-hep1 expression was associated with ERα binding to HepERE but not classical ERE. Taken together, a novel signaling pathway was revealed and the regulatory mechanism associated with the ERα and HepERE element on immunomodulation of OM-hep1 expression upon EE2 exposure was first reported here.A novel regulatory element (HepERE) associated with down-regulation of OM-hep1 expression upon EE2 exposure was discovered.EE2-mediated signal pathway associated with immunomodulation of hepcidin did not share the canonical ER/ERE signal pathway.The new ER/HepERE signal pathway enriched the transcriptional regulatory mechanism of hepcidin expression.