Non-celiac gluten sensitivity (NCGS) and irritable bowel syndrome (IBS) frequently overlap. Although, gluten-free diet (GFD) and low fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAP) improve the IBS clinical picture, many aspects remain unclear. Therefore, we designed a study to evaluate gluten tolerance, anxiety and quality of life in a specific study population. Fifty IBS patients were asked to follow a low FODMAP strict GFD for 6 weeks and were then randomly allocated to the following groups for a further 6 weeks: (A) receiving 8 g/day of gluten for 2 weeks; gluten-tolerating subjects received 16 g/day for 2 weeks and then 32 g/day for a further 2 weeks; (B) continuing to follow a low FODMAP strict GFD; and (C) receiving a gluten-containing diet. After the first 6 weeks, symptom scores significantly improved. Pain severity, bloating and total score were significantly decreased in the GFD and in the high-gluten groups, while the satiety score significantly increased in group C. Between-group analysis revealed significant differences for pain severity (p = 0.02), pain frequency (p = 0.04) and impact on community function (p = 0.02) at the end of the study. Our findings suggest that low FODMAP strict GFD could be prescribed in IBS patients and would reduce anxiety and improve the quality of life.