Although pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates insulin secretion, its net influence on glucose homeostasis in vivo has not been established. We therefore examined the action of PACAP-27 and PACAP-38 on insulin secretion, insulin sensitivity, and glucose disposal as derived from the minimal model of glucose disappearance during an intravenous glucose tolerance test in anesthetized mice. PACAP-27 and PACAP-38 markedly and equipotently potentiated glucose-stimulated insulin secretion, with a half-maximal effect at 33 pmol/kg. After PACAP-27 or PACAP-38 (1.3 nmol/kg), the acute (1–5 min) insulin response was 3.8 ± 0.4 nmol/l (PACAP-27) and 3.3 ± 0.3 nmol/l (PACAP-38), respectively, vs. 1.4 ± 0.1 nmol/l after glucose alone (P < 0.001), and the total area under the curve for insulin (AUC_insulin) was potentiated by 60% (P < 0.001). In contrast, PACAP-27 and PACAP-38 reduced the insulin sensitivity index (S_I) [0.23 ± 0.04 10⁻⁴min⁻¹/(pmol/l) for PACAP-27 and 0.29 ± 0.06 10⁻⁴min⁻¹/(pmol/l) for PACAP-38 vs. 0.46 ± 0.02 10⁻⁴min⁻¹/(pmol/l) for controls (P < 0.01)]. Furthermore, PACAP-27 or PACAP-38 did not affect glucose elimination determined as glucose half-time or the glucose elimination rate after glucose injection or the area under the curve for glucose. Moreover, glucose effectiveness and the global disposition index (AUC_insulin times S_I) were not affected by PACAP-27 or PACAP-38. Finally, when given together with glucose, PACAP-27 did not alter plasma glucagon or norepinephrine levels but significantly increased plasma epinephrine levels. We conclude that PACAP, besides its marked stimulation of insulin secretion, also inhibits insulin sensitivity in mice, the latter possibly explained by increased epinephrine. This complex action explains why the peptide does not enhance glucose disposal.