Endogenous opioids, particularly dynorphins, have been implicated in regulation of energy balance, but it is not known how they mediate this in vivo. We investigated energy homeostasis in dynorphin knockout mice (Dyn(-/-) mice) and probed the interactions between dynorphins and the neuropeptide Y (NPY) system. Dyn(-/-) mice were no different from wild types with regards to body weight and basal and fasting-induced food intake, but fecal output was increased, suggesting decreased nutrient absorption, and they had significantly less white fat and lost more weight during a 24-h fast. The neuroendocrine and thermal responses to fasting were at least as pronounced in Dyn(-/-) as in wild types, and there was no stimulatory effect of dynorphin knockout on 24-h energy expenditure (kilocalories of heat produced) or physical activity. However, Dyn(-/-) mice showed increased circulating concentrations of 3,4-dihydroxyphenlacetic acid and 3,4-dihydroxyphenylglycol, suggesting increased activity of the sympathetic nervous system. The respiratory exchange ratio of male but not female Dyn(-/-) mice was reduced, demonstrating increased fat oxidation. Interestingly, expression of the orexigenic acting NPY in the hypothalamic arcuate nucleus was reduced in Dyn(-/-) mice. However, fasting-induced increases in pre-prodynorphin expression in the arcuate nucleus, the paraventricular nucleus, and the ventromedial hypothalamus but not the lateral hypothalamus were abolished by deletion of Y(1) but not Y(2) receptors. Therefore, ablation of dynorphins results in increases in fatty acid oxidation in male mice, reductions in adiposity, and increased weight loss during fasting, possibly via increases in sympathetic activity, decreases in intestinal nutrient absorption, and interactions with the NPYergic system.