Colonic transit and mucosal integrity are believed to be impaired in obesity. However, a comprehensive assessment of altered colonic functions, inflammatory changes and neuronal signalling of obese animals is missing. In mice, we studied the impact of diet-induced obesity (DIO) on: (i) in vivo colonic transit; (ii) signalling in the myenteric plexus by recording responses to nicotine and 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), together with the expression of tryptophan hydroxylase (TPH) 1 and 2, serotonin reuptake transporter, choline acetyltransferase and the paired box gene 4; and (iii) expression of proinflammatory cytokines, epithelial permeability and density of macrophages, mast cells and enterochromaffin cells. Compared with controls, colon transit and neuronal sensitivity to nicotine and 2-methyl-5-HT were enhanced in DIO mice fed for 12 weeks. This was associated with increased tissue acetylcholine and 5-hydroxytryptamine (5-HT) content, and increased expression of TPH1 and TPH2. In DIO mice, upregulation of proinflammatory cytokines was found in fat tissue, but not in the gut wall. Accordingly, mucosal permeability or integrity was unaltered without signs of immune cell infiltration in the gut wall. Body weight showed positive correlations with adipocyte markers, tissue levels of 5-HT and acetylcholine, and the degree of neuronal sensitization. DIO mice fed for 4 weeks showed no neuronal sensitization, had no signs of gut wall inflammation and showed a smaller increase in leptin, interleukin-6 and monocyte chemoattractant protein 1 expression in fat tissue. DIO is associated with faster colonic transit and impacts on acetylcholine and 5-HT metabolism with enhanced responsiveness of enteric neurones to both mediators after 12 weeks of feeding. Our study demonstrates neuronal plasticity in DIO prior to the development of a pathological histology or abnormal mucosal functions. This questions the common assumption that increased mucosal inflammation and permeability initiate functional disorders in obesity.