The now banned anorectic molecule, dexfenfluramine, promotes serotonin release through a serotonin transporter-dependent mechanism and has been widely prescribed for the treatment of obesity. Previous studies have identified that 5-HT2B receptors play important roles in dexfenfluramine-side effects, i.e. pulmonary hypertension, plasma serotonin level regulation, and valvulopathy. We thus investigated a putative contribution of 5-HT2B receptors in dexfenfluramine-dependent feeding behavior in mice. Interestingly, the hypophagic response to dexfenfluramine (3-10 mg/kg) observed in wildtype mice (1-4 hours) was eliminated in mice lacking 5-HT2B receptors (5-HT2B-/-). These findings were further validated by the lack of hypophagic response to dexfenfluramine in wildtype mice treated with RS127445, a highly selective and potent antagonist (pKi=8.22±0.24). By microdialysis, we observed that in 5-HT2B-/- awake mice, the dexfenfluramine-induced hypothalamic peak of serotonin release (1 hour) was strongly reduced (4 fold) compared to wildtype. Moreover, using hypothalamic synaptosomes, we established the serotonergic neurons autonomous properties of this effect: a strong serotonin release was observed upon dexfenfluramine stimulation of synaptosomes preparation from wildtype but not from mice lacking active 5-HT2B receptors. These findings strongly suggest that activation of presynaptic 5-HT2B receptors is a limiting step in the serotonin transporter dependant-releasing effect of dexfenfluramine, while other serotonin receptors act downstream with respect to feeding behavior.