For decades, the gut was thought to play an important role in sepsis pathogenesis. Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Organ failure assessment for sepsis focuses on respiratory, cardiovascular, hepatic, renal, neurological, and haematological systems. Unfortunately, symptoms of gut failure are non-specific and are therefore not assessed. The composition of the intestinal microbiome, however, is affected by sepsis, and might contribute to the development of organ failure. Experimental work underscores the role of the microbiota in maintaining gut-barrier function, and modulation of the innate and adaptive immune system. Translation of these preclinical findings into functional characterisations will be essential to understand how disruption of commensals affects susceptibility and outcome of sepsis. In this Review, we identify knowledge gaps which, if addressed, will help researchers understand the role of the microbiota in sepsis, and provide microbiota-targeted tools to improve sepsis management.