Technical Abstract: Objective: These studies describe the in vivo metabolic actions of the novel amylin mimetic peptide davalintide (AC2307) in rodents, and compare these effects to those of the native peptide. Research Design and Methods: The anti-obesity effects of davalintide were examined following intraperitoneal injection or sustained peripheral infusion via subcutaneously implanted osmotic pumps. The effect of davalintide on food intake following lesion of the area postrema (AP), and neuronal activation as measured by c-Fos, were also investigated. Results: Similar to amylin, davalintide bound with high affinity to amylin, calcitonin, and calcitonin gene-related peptide receptors. Acutely, davalintide displayed greater suppression of dark-cycle feeding and an extended duration of action compared to amylin (23 versus 6 h). Davalintide had no effect on locomotor activity or kaolin consumption at doses that decreased food intake. Davalintide-induced weight loss via infusion was dose-dependent, durable up to 8 weeks, fat-specific and lean-sparing, and associated with a shift in food preference away from high-fat (palatable) chow. Metabolic rate was maintained during active weight loss. Both davalintide and amylin failed to suppress food intake following lesion of the AP and activated similar brain nuclei, with davalintide displaying an extended duration of c-Fos expression compared to amylin (8 versus 2 h). Conclusion: Davalintide displayed enhanced in vivo metabolic activity over amylin while retaining the beneficial properties possessed by the native molecule. In vitro receptor binding, c-Fos expression, and AP lesion studies suggest the metabolic actions of davalintide and amylin occur through activation of similar neuronal pathways.