Objective: To compare the incidence of multiple sclerosis (MS) among women who had undergone assisted reproductive technology (ART) treatment with the women who had conceived a child without previous ART treatment. Design: A register-based nationwide cohort study. Patient(s): Women with a first ovarian stimulation cycle before in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (i.e., ART treatment) recorded in the Danish IVF register between 1996 and 2018; and women recorded in the Danish Medical Birth Register with the birth of their first child where date of conception is between 1996 and 2018. The cohort was observed until March 10, 2021. Intervention(s): Mainly included IVF, ICSI, and fresh embryo transfer with hormone stimulation. Main Outcome Measures: A diagnosis of MS recorded in the Danish Multiple Sclerosis Registry. Crude and adjusted hazard ratios (aHRs) with 95% confidence intervals (CIs) were calculated. Results: A total of 585,716 women were included in the cohort of which 63,791 (11%) were exposed to at least one initiated IVF or ICSI cycle during the study period. Cycles with oocyte donation were excluded. The median follow-up time for the entire cohort was 12.4 years (Q1–Q3= 6.6–18.1). Compared with women conceiving without previous ART, ART treated women were older (31.8 years vs. 27.5 years), more often had a university degree (45% vs. 36%), and more often had received other fertility treatments than IVF or ICSI before cohort entry (26% vs. 3%). We found no association between incident MS and exposure to ART compared with non-ART pregnancy (aHR=1.08; 95 % CI, 0.93–1.25). An analysis following intention-to-treat principle on a propensity score matched sub cohort confirmed our results. In subgroup analysis including all ART cycles among the ART treated women, we found no increased risk of MS within 2 years of ART cycle start for successful ART cycles (pregnancy) compared with failed ART cycles (no pregnancy) (aHR=1.01; 95% CI, 0.58–1.76). We found a non-significant trend toward increased risk of MS with increasing numbers of ART cycles although based on small numbers. Conclusion(s): Women treated with ART do not seem to be at increased risk of developing MS compared with the women not exposed to ART.