To investigate the effects of dietary arachidonic acid (ARA) on the sex steroid hormone production and the gene expressions of the key proteins in steroidogenesis in gonads of turbot Scophthalmus maximus broodstocks, three isonitrogenous and isolipidic (lipid content 13%) experimental diets were formulated to contain different ARA levels: the control diet without ARA supplementation (C, 0.72% ARA of total fatty acids) and two diets with low (5.63% of total fatty acids, ARA-L) or high ARA (15.03% of total fatty acids, ARA-H) level. The experimental diets were randomly assigned to 9 tanks of 3-year-old turbot (25 fish in each tank). Fish were reared in an indoor flowing seawater system and fed to apparent satiation twice daily. At the end of the feeding trial, sex steroid hormone production was assayed and gonad samples from females and males were collected to determine the gene expressions of sex steroid-synthesizing proteins. The results showed that compared to the control group, ARA-H significantly reduced the estradiol production in females but in males ARA-L significantly reduced the testosterone production. In ovaries, ARA-H significantly reduced the mRNA expression of follicle stimulating hormone receptor, but ARA-L significantly increased the mRNA expression of 17α-hydroxylase. In testes, ARA-L significantly reduced the mRNA expression of steroidogenic acute regulatory protein and 17α-hydroxylase, but significantly increased the mRNA expression of aromatase. Dietary ARA supplementation increased the ARA accumulation but decreased the EPA accumulation in fish tissues. The ARA concentration in ovaries was higher than that in testes. In conclusion, certain content of dietary ARA inhibited the estradiol and testosterone production in immature turbot broodstock. In ovaries, this inhibition may be attributed to the inhibition on follicle stimulating hormone receptor, but in testes it may be attributed to the inhibition on steroidogenic acute regulatory protein and 17α-hydroxylase.