Ghrelin exists in two forms in circulation, acyl ghrelin and des-acyl ghrelin, both of which have distinct and fundamental roles in a variety of physiological functions. Despite this fact, a large proportion of papers simply measure and refer to plasma ghrelin without specifying the acylation status. It is therefore critical to assess and state the acylation status of plasma ghrelin in all studies. In this study we tested the effect of des-acyl ghrelin administration on the hypothalamic-pituitary-adrenal axis and on anxiety-like behavior of mice lacking endogenous ghrelin and in ghrelin-O-acyltransferase (GOAT) knockout (KO) mice that have no endogenous acyl ghrelin and high endogenous des-acyl ghrelin. Our results show des-acyl ghrelin produces an anxiogenic effect under nonstressed conditions, but this switches to an anxiolytic effect under stress. Des-acyl ghrelin influences plasma corticosterone under both nonstressed and stressed conditions, although c-fos activation in the paraventricular nucleus of the hypothalamus is not different. By contrast, GOAT KO are anxious under both nonstressed and stressed conditions, although this is not due to corticosterone release from the adrenals but rather from impaired feedback actions in the paraventricular nucleus of the hypothalamus, as assessed by c-fos activation. These results reveal des-acyl ghrelin treatment and GOAT deletion have differential effects on the hypothalamic-pituitary-adrenal axis and anxiety-like behavior, suggesting that anxiety-like behavior in GOAT KO mice is not due to high plasma des-acyl ghrelin.