Disruption of the pituitary adenylate cyclase-activating polypeptide (PACAP) gene in mouse has demonstrated a role for this highly conserved neuropeptide in the regulation of metabolism and temperature control. Localization of PACAP neurons within hypothalamic nuclei that regulate appetite suggest PACAP may affect feeding and thus energy balance. We utilized PACAP-null mice to address this question, examining both food intake and energy expenditure. PACAP-null mice were leaner than wild-type littermates due to decreased adiposity and displayed increased insulin sensitivity. The lean phenotype in the PACAP-null mice was completely eliminated if animals were fed a high fat diet or housed near thermoneutrality (28C). Further metabolic analyses of PACAP-null mice housed at 21C indicated that the reduced body weight could not be explained by decreased food intake, increased metabolic rate or increased locomotor activity. The thyroid hormone axis of PACAP-null mice was affected, as mRNA levels of hypothalamic TRH and brown adipose tissue type 2 deiodinase were reduced in PACAP-null mice housed at room temperature, and brain deiodinase activity was lower in PACAP-null mice after an acute cold challenge compared with wild-type controls. These results demonstrate that PACAP is not required for the regulation of food intake yet is necessary to maintain normal energy homeostasis likely playing a role in central cold-sensing mechanisms.