A recent study demonstrated that 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) may have an adverse effect on the reproduction in marine medaka (Oryzias melastigma), but the molecular mechanisms remain largely unknown. In this study, we investigated the protein expression profiles of male and female gonads of O. melastigma exposed to dietary BDE-47 at two dosages (0.65 and 1.30 μg/g/day, respectively) for 21 days. Extracted proteins were labeled with iTRAQ and analyzed on a MALDI TOF/TOF analyzer, as results, 133 and 144 unique proteins were identified in testis and ovary, respective, and they exerted dose- and sex-dependent expression patterns. In testis, among the 42 differentially expressed proteins; down-regulation of histone variants and parvalbumins implicated BDE-47 may disrupt the spermatogenesis and induce sterility in fishes. In ovary, 38 proteins were differentially expressed; the elevation of vitellogenins and apolipoprotein A–I expression indicated BDE-47 acts as an estrogen-mimicking compound and led to reproductive impairment in O. melastigma.