The Ts65Dn segmental mouse model of Down syndrome (DS) possesses a triplication of the section of chromosome 16 that is most homologous to the human chromosome 21 that is trisomic in DS. This model exhibits many of the characteristics of DS including small size, developmental delays, and a decline of cholinergic systems and cognitive function with age. Recent studies have shown that vasoactive intestinal peptide (VIP) systems are upregulated in aged Ts65Dn mice and that VIP dysregulation during embryogenesis is followed by the hypotonia and developmental delays as seen in both DS and in Ts65Dn mice. Additionally, astrocytes from aged Ts65Dn brains do not respond to VIP stimulation to release survival-promoting substances. To determine if VIP dysregulation is age-related in Ts65Dn mice, the current study examined VIP and VIP receptors (VPAC-1 and VPAC-2) in postnatal day 8 Ts65Dn mice. VIP and VPAC-1 expression was significantly increased in the brains of trisomic mice compared with wild-type mice. VIP-binding sites were also significantly increased in several brain areas of young Ts65Dn mice, especially in the cortex, caudate/putamen, and hippocampus. Further, in vitro treatment of normal neurons with conditioned medium from VIP-stimulated Ts65Dn astrocytes from neonatal mice did not enhance neuronal survival. This study indicates that VIP anomalies are present in neonatal Ts65Dn mice, a defect occurs in the signal transduction mechanism of the VPAC-1 VIP receptor, cortical astrocytes from neonatal brains are dysfunctional, and further, that VIP dysregulation may play a significant role in DS.