Background. Mounting evidence points towards a pivotal role of gut microbiota in multiple sclerosis (MS) pathophysiology. Yet, if disease-modifying treatments alter microbiota composition and whether microbiota shape treatment response and side-effects remains unclear. Methods. In this prospective observational pilot study, we assessed the effect of dimethyl fumarate (DMF) on gut microbiota and on host/microbial metabolomics in a cohort of 20 MS patients. Results. Combining of state-of-the-art microbial sequencing, metabolome mass spectrometry and computational analysis, we identified longitudinal changes of gut microbiota composition under DMF-treatment and an increase in citric acid cycle metabolites. Notably, DMF-induced lymphopenia, a clinically relevant safety concern, correlated with distinct baseline microbiome signatures in MS patients. Discussion. We identified gastrointestinal microbiota as a key therapeutic target of metabolic properties of DMF. By characterizing gut microbial composition as a candidate risk factor for DMF-induced lymphopenia, we provide novel insights into the role of microbiota in mediating clinical side-effects.