Recent evidence that 5-hydroxytryptamine (5-HT or serotonin) enhances the release and the gene expression of vasopressin and oxytocin in the hypothalamic paraventricular nucleus (PVN) suggests that 5-HT can excite the PVN magnocellular neurons. The objective of this study was to examine the underlying mechanisms for such excitatory action in the electrophysiologically identified hypothalamic PVN magnocellular neurons in rats using whole-cell patch-clamp. We found that 5-HT weakly depolarizes 33.3% of PVN magnocellular neurons in the presence of tetrodotoxin. A minuscule inward current was produced by 5-HT in 48% of the cells, which was attenuated when the 5-HT4 antagonist GR113808 or the 5-HT7 antagonist SB269970 was added. In addition, 5-HT reduced the frequency of miniature inhibitory postsynaptic currents in a dose-dependent manner. This inhibition was mimicked by the 5-HT1B agonist CP93129, and reversed in the presence of 5-HT1B antagonists cyanopindolol and SB224289. Besides, 5-HT induced a biphasic effect on the frequency of miniature excitatory postsynaptic currents, comprising a transient inhibition and a delayed concentration-dependent excitation (onset latency ∼ 5 min). The facilitation was mimicked by the 5-HT2A/2C agonist DOI and abolished in the presence of the 5-HT2C antagonist RS102221. Our findings reveal that 5-HT directly increases the excitability of the PVN magnocellular neurons via multiple receptor subtypes and mechanisms. This may help understanding the regulation of 5-HT-induced hormone release and feeding behavior in the PVN.