Background: Irritable bowel syndrome (IBS) is a functional gastrointestinal disease of unknown etiology. Researchers have recently drawn attention to the possible role of viruses in the development of IBS and provided evidence in this regard. In this study, it was decided to investigate the possible role of rotavirus infection in the onset of IBS. Methods: Stool and serum samples were collected from 40 patients with IBS and 40 healthy individuals. To evaluate the previous exposure to rotavirus we checked the presence and concentration of anti-rotavirus IgG by ELISA. ELISA test was performed on the serum samples. A real-time polymerase chain reaction (PCR) test was also used to measure the viral load in the stool. Finally, the data were analyzed by SPSS version 22 software. Results: No significant relationship was found between anti-rotavirus IgG presence and its level in the serum of case and healthy individuals (P value>0.05). Moreover, there was no significant difference between the viral genome load in the stool samples of the two groups (P value>0.05). Conclusion: According to the results, it seems unlikely that a link exists between rotavirus infection and the onset of IBS, but the possible role of other gastrointestinal viruses, including coronavirus, remains.