The incretin, glucagon-like peptide-1 (GLP-1) is a major player in the gut-brain axis regulation of energy balance and in fish it seems to exert a negative influence on food intake. In this study, we investigated the role of the brain serotonergic system in the effects promoted by a peripheral GLP-1 injection on food intake in rainbow trout (Oncorhynchus mykiss). For this, in a first experiment the incretin was intraperitoneally injected (100 ng/g body weight) alone or in combination with a 5HT receptor antagonist (SB 242084, 1 µg/g body weight) and food intake was measured 30, 90, and 180 min later. In a second experiment, we studied the effect of these treatments on mRNA abundance of hypothalamic neuropeptides that control food intake. In addition, the effect of GLP-1 on serotonin metabolism was assessed in hindbrain and hypothalamus. Our results show that GLP-1 induced a significant food intake inhibition, which agreed with the increased expression of anorexigenic neuropeptides pomc and cart in the hypothalamus. Furthermore, GLP-1 stimulated the synthesis of serotonin in the hypothalamus, which might be indicative of a higher use of the neurotransmitter. The effects of GLP-1 on food intake were partially reversed when a serotonin receptor antagonist, SB 242084, was previously administered to trout. This antagonist also reversed the stimulatory effect of the hormone in hypothalamic pomca1 mRNA abundance. We conclude that hypothalamic serotonergic pathways are essential for mediating the effects of GLP-1 on food intake in rainbow trout. In addition, the 5HT2C receptor subtype seems to have a prominent role in the inhibition of food intake induced by GLP-1 in this species.