Neurotensin (NT) is a gastro-intestinal hormone involved in several pathways that regulate energy and glucose homeostasis. NT was hypothesized to act in synergy with incretin hormones to potentiate its anti-diabetic effects. Additionally, circulating NT levels were shown to rise after bariatric surgery-induced weight loss. Knowledge of NT-secreting cells distribution along the small intestine and its variation according to diabetes status could provide insights on NT role in mediating type 2 diabetes (T2D) improvement after bariatric surgery. So, our aims were to characterize NT-expressing cell distribution along the human small intestine and to compare the relative density of NT-expressing cells in the small intestine of individuals with and without T2D undergoing bariatric surgery for obesity treatment. Autopsy-derived small intestine fragments (n = 30) were obtained at every 20 cm along the entire intestinal length. Additionally, jejunum biopsies (n = 29) were obtained during elective gastric bypass interventions from patients with (n = 10) or without T2D (n = 18). NT-expressing cells were identified by immunohistochemistry and quantified via computerized morphometric analysis. NT-expressing cell density increased along the human small intestine. NT-expressing cell density was significantly higher from 200 cm distal to the duodenojejunal flexure onward, as well as in subjects with T2D when compared to those without T2D. NT-expressing cell density increases along the human small gut, and a higher density is found in individuals with T2D. This finding suggests a potential role for NT in the mechanisms of disease and T2D improvement observed after bariatric surgery.