Although amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibitory action on neuropeptide Y (NPY), an appetite neurotransmitter abundant in the brain, molecular mechanisms underlying this effect are not well known. This manuscript examined the possible role of protein kinase A (PKA) and cAMP response element binding protein (CREB) signaling in this anorectic effect, and the results showed that both PKA and CREB mRNA levels in hypothalamus were increased following AMPH treatment, which were relevant to a reduction of NPY mRNA level. To determine if PKA or CREB was involved in the anorectic response, the intracerebroventricular infusions of antisense oligonucleotide (or missense control) were performed at 60 min before daily AMPH treatment in conscious rats, and results showed that either PKA or CREB knock down could block AMPH-induced anorexia as well as restore NPY mRNA level, indicating the respective involvement of PKA and CREB signaling in the regulation of NPY gene expression. It is suggested that hypothalamic PKA and CREB signaling may involve the central regulation of AMPH-mediated feeding suppression via the modulation of NPY gene expression.