Background: Gastrointestinal dysfunction is very common in diabetic patients. We assessed the changes in the colonic enteric nervous system using colectomy specimens and intestinal biopsies from diabetic subjects and age-matched controls. Methods: In control and diabetic colons, we determined the total ganglion area (hematoxylin-eosin staining), changes in neuronal markers-PGP 9.5, peripherin, nNOS, NPY, ChAT and VIP (by immunostaining), apoptosis (cleaved caspase-3 staining) and reduced glutathione levels (HPLC). Superoxide dismutase (SOD) mRNA was determined in enteric ganglia isolated by laser capture micro dissection. Isometric muscle recording was used to assess contraction and relaxation responses of colonic circular muscle strips. Apoptosis in enteric neurons under hyperglycemia in vitro was determined by cleaved caspase-3 Western blotting and protective effects of lipoic acid were evaluated. Key Results: Diabetic subjects had higher incidence of lower gastrointestinal symptoms like constipation and diarrhea at baseline prior to surgery. Diabetic ganglia displayed significant decrease in ganglion size due to enhanced apoptosis and loss of peripherin, nNOS, NPY and ChAT neurons. Reduced glutathione levels in the diabetic colon (HbA1C>7%) were significantly less than the control, indicating increased oxidative stress. Colonic circular muscle strips from diabetic subjects showed impaired contraction and relaxation responses compared to the healthy controls. Hyperglycemia-induced cleaved caspase-3 in enteric neurons was reversed by lipoic acid. Conclusions: Our data demonstrate loss of enteric neurons in the colon due to increased oxidative stress and apoptosis which may cause the motility disturbances seen in human diabetes. Antioxidants may be of therapeutic value for preventing motility disorders in diabetes.