Many patients with irritable bowel syndrome (IBS) perceive that their symptoms are triggered by wheat-containing foods. We assessed symptoms and gastrointestinal transit before and after a gluten-free diet (GFD) in unselected patients with IBS and investigated biomarkers associated with symptoms.We performed a prospective study of 50 patients with IBS (ROME III, all subtypes), with and without serologic reactivity to gluten (anti-gliadin IgG and IgA), and 25 healthy subjects (controls) at a university hospital in Hamilton, Ontario, Canada between 2012-2016. Gastrointestinal transit, gut symptoms, anxiety, depression, somatization, dietary habits, and microbiota composition were studied before and after 4 weeks of a GFD. HLA-DQ2/DQ8 status was determined. GFD compliance was assessed by a dietitian and by measuring gluten peptides in stool.There was no difference in symptoms among patients at baseline, but after the GFD, patients with anti-gliadin IgG and IgA reported less diarrhea than patients without these antibodies (P=.03). Compared with baseline, IBS symptoms improved in 18/24 patients (75%) with anti-gliadin IgG and IgA and in 8/21 patients (38%) without the antibodies. Although constipation, diarrhea, and abdominal pain were reduced in patients with anti-gliadin IgG and IgA, only pain decreased in patients without these antibodies. Gastrointestinal transit normalized in a higher proportion of patients with anti-gliadin IgG and IgA. Anxiety, depression, somatization, and well-being increased in both groups. The presence of anti-gliadin IgG was associated with overall reductions in symptoms (adjusted odds ratio compared to patients without this antibody, 128.9; 95% CI, 1.16-1427.8; P=.04). Symptoms were reduced even in patients with anti-gliadin IgG and IgA who reduced gluten intake but were not strictly compliant with the GFD. In controls, a GFD had no effect on gastrointestinal symptoms or gut function.Anti-gliadin IgG can be used as a biomarker to identify patients with IBS who might have reductions in symptoms, particularly diarrhea, on a GFD. Larger studies are needed to validate these findings.