Irritable Bowel Syndrome (IBS) is one of the most commonly diagnosed gastrointestinal disorders, and its etiology is believed to be multifactorial. The role of smoking in the pathophysiology of IBS still remains inconclusive. Hence, we aim to investigate whether or not an association exists between smoking and IBS. A cross-sectional study was conducted in a tertiary care hospital of Karachi. A sample size of 200 smokers and 200 non smokers was selected. A two-part self-reported questionnaire was administered to the participants and the diagnosis of IBS was based on the Rome III diagnostic criteria. All data were analyzed using the Statistical Package for the Social Sciences version 17. Statistical tests employed were Independent samples t-test, chi-square test, and binary logistic regression analysis to obtain Odds Ratio (OR) for various variables. A P-value <0.05 was considered statistically significant. Overall prevalence of IBS based on Rome III criteria was 9.5% (95% CI 9.45-9.60). Smoking was significantly found to be associated with IBS (P=0.002). Binary logistic regression analysis indicated that non-smokers were more likely to have IBS (Crude OR: 3.00, 95% CI 1.23-7.30) than smokers, and females were more likely to have IBS (Crude OR: 3.73, 95% CI 1.56-7.69) than males. Age, BMI, and food groups were not found to be significantly associated with IBS. Our study concludes that IBS was more prevalent among non-smokers and amongst females. Further studies are required to firmly establish the role of smoking in IBS.© 2020 Iran University of Medical Sciences.