Anorexia nervosa is an eating disorder often associated with intestinal disorders. To explore the underlying mechanisms of these disorders, the colonic proteome was evaluated during activity‐based anorexia. Female C57Bl/6 mice were randomized into three groups: Control, Limited Food Access (LFA) and Activity‐Based Anorexia (ABA). LFA and ABA mice had a progressive limited access to food but only ABA mice had access to an activity wheel. On colonic mucosal protein extracts, a 2D PAGE‐based comparative proteomic analysis was then performed and differentially expressed proteins were identified by LC‐ESI‐MS/MS. Twenty‐seven nonredundant proteins that were differentially expressed between Control, LFA, and ABA groups were identified. ABA mice exhibited alteration of several mitochondrial proteins involved in energy metabolism such as dihydrolipoyl dehydrogenase and 3‐mercaptopyruvate sulfurtransferase. In addition, a downregulation of mammalian target of rapamycin (mTOR) pathway was observed leading, on the one hand, to the inhibition of protein synthesis, evaluated by puromycin incorporation and mediated by the increased phosphorylation of eukaryotic elongation factor 2, and on the other hand, to the activation of autophagy, assessed by the increase of the marker of autophagy, form LC3‐phosphatidylethanolamine conjugate/Cytosolic form of Microtubule‐associated protein 1A/1B light chain 3 (LC3II/LC3I) ratio. Colonic mucosal proteome is altered during ABA suggesting a downregulation of energy metabolism. A decrease of protein synthesis and an activation of autophagy were also observed mediated by mTOR pathway.