Gastrointestinal (GI) symptoms are common in patients with diabetes mellitus (DM). The electromagnetic 3D-Transit system allows assessment of regional transit times and motility patterns throughout the GI tract. We aimed to compare GI transit times and detailed motility patterns of the colon in patients with DM and GI symptoms to those of healthy controls (HC). We further aimed to determine whether any abnormalities in motility were reversible by cholinergic stimulation. We compared 18 patients with DM with 20 HC by means of the 3D-Transit system. Patients were studied before and during oral administration of 60mg pyridostigmine. Compared to HC, patients had prolonged gastric emptying (DM: 3.3hours (interquartile range (IQR) 2.6-4.6); HC: 2.3hours (IQR 1.7-2.7) (P<.01)), colonic transit time (DM: 52.6hours (IQR 23.3-83.0); HC: 22.4hours (IQR 18.9-43.6) (P=.02)), and whole gut transit time (DM: 69.4hours (IQR 32.9-103.6); HC: 30.3hours (IQR 25.2-49.9) (P<.01)). In addition, compared to HC, patients had prolonged transit time in the ascending colon (DM: 20.5hours (IQR 11.0-44.0); HC: 8.0hours (IQR 3.8-21.0) (P<.05)) and more slow retrograde movements in the colon (DM: 2 movements (IQR 1-4); HC: 1 movement (IQR 0-1) (P=.01)). In patients, pyridostigmine increased the number of bowel movements (P<.01) and reduced small intestine transit times (P<.05). Patients with DM and GI symptoms have longer than normal GI transit times. This is only partly reversible by pyridostigmine. The increased number of retrograde colonic movements in patients could potentially explain the abnormally long transit time in proximal colon.