There are few if any reports regarding the role of lifetime waterpipe smoking in the etiology of multiple sclerosis (MS). In a population-based incident case-control study conducted in Tehran, Iran, we investigated the association between waterpipe smoking and MS, adjusted for confounders. Cases (n = 547) were patients aged 15-50 years identified from the Iranian Multiple Sclerosis Society between 2013 and 2015. Population-based controls (n = 1,057) were persons aged 15-50 years recruited through random digit telephone dialing. A doubly robust estimation method, the targeted maximum likelihood estimator (TMLE), was used to estimate the marginal risk ratio and odds ratio for the association between waterpipe smoking and MS. The estimated risk ratio and odds ratio were both 1.70 (95% confidence interval: 1.34, 2.17). The population attributable fraction was 21.4% (95% confidence interval: 4.0, 38.8). Subject to the limitations of case-control studies in interpreting associations causally, these results suggest that waterpipe use, or strongly related but undetermined factors, increases the risk of MS. Further epidemiologic studies, including nested case-control studies, are needed to confirm these findings.