Introduction: Irritable bowel syndrome (IBS) is a functional gastrointestinal condition, in which patients suffer from abdominal pain, bloating, and change in bowel habits. Some researchers examined the potential therapeutic role of vitamin D3 (Cholecalciferol) to alleviate symptoms of IBS, however, the outcomes of their studies were controversial. Objective: to assess the effect of vitamin D3 supplement on gastrointestinal symptoms in patients suffering from irritable bowel syndrome. Methods: a double-blinded, randomized, placebo-controlled trial. Eighty patients with IBS attending the family practice center in El-Mahsama, Ismailia, Egypt were recruited, after applying inclusion and exclusion criteria. The study participants were allocated randomly into two groups, (1) the intervention and (2) the control group. The intervention group received 4000 IU of vitamin D3 orally and the control group received a placebo of edible paraffin orally. Both groups received their assigned treatment daily for 12 weeks. Irritable bowel syndrome symptom severity score (IBSSSS) was assessed at baseline and after 12 weeks. Results: IBSSSS was significantly reduced in the intervention group (114.36 ± 67.36) after 12 weeks compared to the control group (292.13 ± 74.77) (p < 0.001). Regarding IBSSSS difference, patients receiving vitamin D were found to have significantly higher score difference (-164.72 ± 67.77) than participants who took the placebo (-12.13 ± 50.78) (p < 0.001).Conclusion: Patients suffering from IBS felt better and their gastrointestinal symptoms improved after taking vitamin D3 supplementation.