We investigated the effects of fluoxetine, a selective serotonin reuptake inhibitor, on neuroendocrine function and the reproductive axis in female goldfish. Fish were given intraperiteonal injections of fluoxetine twice a week for 14 days, resulting in five injections of 5 µg fluoxetine/g of body weight. We measured the monoamine neurotransmitters serotonin, dopamine and norepinephrine in addition to their metabolites using HPLC. Homovanillic acid, a metabolite in the dopaminergic pathway, increased significantly in the hypothalamus. Plasma estradiol levels were measured by radioimmunoassay and were significantly reduced approximately 3-fold after fluoxetine treatment. We found that fluoxetine also significantly reduced the expression of ER1 mRNA by 4-fold in both the hypothalamus and telencephalon and ER mRNA in the telencephalon by 1.7-fold. Fluoxetine had no effect on the expression of ER2 mRNA in the hypothalamus or telencephalon. Microarray analysis identified isotocin, a neuropeptide that stimulates reproductive behaviour in fish, as a candidate gene affected by fluoxetine treatment. Real-time RT-PCR verified that isotocin mRNA was down-regulated approximately 6-fold in the hypothalamus and 5-fold in the telencephalon. Intraperitoneal injection of isotocin (1 µg /g) increased plasma estradiol, providing a potential link between changes in isotocin gene expression with decreased circulating estrogen in fluoxetine-injected fish. Our results reveal targets of serotonergic modulation in the neuroendocrine brain and indicate that fluoxetine has the potential to affect sex hormones and modulate genes involved in reproductive function and behaviour in the brain of female goldfish. We discuss these findings in the context of endocrine disruption because fluoxetine has been detected in the environment.