Serotonin [or 5-hydroxytryptamine or (5-HT)] has been implicated as a key modulator in energy homeostasis and a primary focus in the treatment of obesity. There is growing evidence that 5-HT, acting through the 5-HT 1B receptor (5-HT(1B)R) in the paraventricular nucleus of the hypothalamus (PVN), is important to this regulation. However, there is some contention as to whether 5-HT(1B)R action occurs directly on PVN neurons or indirectly via inhibitory inputs into the PVN. To address these questions, we used a novel clonal, hypothalamic neuronal cell model, adult mouse hypothalamic-2/30 (mHypoA-2/30), expressing a PVN-specific marker, single-minded homolog 1, as well as a complement of PVN neuropeptides, including TRH, vasopressin, ghrelin, nucleobindin-2, and galanin. Adult mouse hypothalamic-2/30 neurons were also found to express the 5-HT(1B)R and 5-HT 6 receptor, but not 2C, all previously linked to feeding regulation. Direct serotonergic stimulation (100 nm to 10 μm) of these neurons resulted in dose-dependent cFos activation. 5-HT (10 μm) suppressed forskolin-induced cAMP levels and induced a rise in intracellular Ca(2+) through ER Ca(2+) release, effects that were mimicked by the 5-HT(1B)R agonists, CGS12066B and CP93129, and that were attenuated in the presence of the 5-HT(1B)R-specific inhibitors, GR55562 and isamoltane hemifumarate. Modest transcriptional changes in ghrelin and nucleobindin-2 were also observed in response to 100 nm and 10 μm 5-HT, respectively. These findings support the model wherein 5-HT action through the 1B receptor subtype occurs directly on PVN neurons, leading to potential modification of neuronal transcriptional and secretory machinery.