Since the three last decades, modifications of lifestyles induce worldwide increase of obesity leading to chronic diseases such as type 2 diabetes mellitus (T2DM) and cardiovascular illnesses which nowadays represent huge public health issues. Currently, the characterization of bioactive molecules isolated from or contained in natural agro/food products to be used as smart food or nutraceuticals represent a promising alternative to chemical drugs in the prevention and the management of obesity-related diseases. In this study, a fish byproduct hydrolysate (FBPH) and its raw material (FBP) were submitted to an in vitro simulated gastrointestinal digestion to further study their ability to modulate gut hormone secretion implicated in food intake regulation and glucose metabolism. Thus, the stimulation of cholecystokinin (CCK) and glucagon like-peptide 1 (GLP-1) secretions following the contact with FBPH and FBP digests were studied using STC-1 enteroendocrine cells. Their ability to inhibit the intestinal DPP-IV activity, a serine peptidase who drastically reduces the GLP-1 activity, was also measured in parallel using Caco-2 enterocyte model. Further we studied the effects of a daily oral administration of FBPH in rats receiving a high-fat diet on glucose and food intake regulations. In vitro results showed the ability of FBPH to highly stimulate CCK and GLP-1 secretion and to inhibit intestinal DPP-IV activity. These results were supported with those obtained in vivo which evidenced that FBPH preload administration was able to decrease short term food intake in high-fat diet fed rats. These very promising results open good perspective to the use of this FBPH as food bioactive to prevent obesity and T2DM.