The disruption in glucose homeostasis characteristic of diabetes and obesity is due partly to an increase in glucose production (GP). However, the mechanisms underlying the regulation of GP in healthy and obese/diabetic settings remain to be elucidated. A duodenal Intralipid infusion activates a duodenal PKC-δ - cholecystokinin (CCK) - CCK1 receptor - PKA neuronal signaling cascade to lower GP. Intralipid is an emulsion of fatty acids containing the highest percentage of linoleic acid (LA; polyunsaturated fatty acid) and oleic acid (OA; monounsaturated fatty acid). The relative ability of individual duodenal fatty acids to regulate GP is currently unknown, as are the potential mechanisms. Therefore, we investigated whether LA and OA lower GP when infused into the duodenum and whether they utilize similar mechanisms as Intralipid in rats and mice in vivo. First, anintraduodenal infusion of OA or LA lowered GP during the pancreatic (basal insulin) euglycemic clamps. Second, co-infusion of duodenal OA with the CCK1 receptor inhibitor, MK-329, abolished the ability of OA to lower GP. Interestingly, co-infusion of duodenal LA with MK-329 did not block the ability of LA to lower GP. Third, anintraduodenal OA or LA infusion failed to lower GP when co-infused with the anesthetic tetracaine. Fourth, acute high fat feeding resulting in early onset obesity, disrupted the ability of duodenal OA or LA infusion to lower GP. In summary, duodenal LA and OA sensing lower GP via a CCK-independent and dependent neuronal network in rats and mice. Dissecting duodenal OA and LA signaling will unveil dietary and/or pharmacological strategies to activate duodenal signaling that lower GP in diabetes and obesity.