Apelin, a recently isolated neuropeptide that is expressed in the supraoptic and the paraventricular nuclei, acts on specific receptors located on vasopressinergic neurons. The increased phasic pattern of these neurons facilitates sustained antidiuresis during dehydration or lactation. Here, we investigated whether apelin interacts with arginine vasopressin (AVP) to maintain body fluid homeostasis. We first characterized the predominant molecular forms of endogenous hypothalamic and plasma apelin as corresponding to apelin 13 and, to a lesser extent, to apelin 17. We then demonstrated that, in lactating rats, apelin was colocalized with AVP in supraoptic nucleus magnocellular neurons and given intracerebroventricularly inhibited the phasic electrical activity of AVP neurons. In lactating mice, intracerebroventricular administration of apelin 17 reduced plasma AVP levels and increased diuresis. Moreover, water deprivation, which increases systemic AVP release and causes depletion of hypothalamic AVP stores, decreased plasma apelin concentrations and induced hypothalamic accumulation of the peptide, indicating that AVP and apelin are conversely regulated to facilitate systemic AVP release and suppress diuresis. Opposite effects of AVP and apelin are likely to occur at the hypothalamic level through autocrine modulation of the phasic electrical activity of AVP neurons. Altogether, these data demonstrate that apelin acts as a potent diuretic neuropeptide counteracting AVP actions through inhibition of AVP neuron activity and AVP release. The coexistence of apelin and AVP in magnocellular neurons, their opposite biological effects, and regulation are likely to play a key role for maintaining body fluid homeostasis.