Objective The gut microbiota is essential for the development of the intestinal immune system. Animal models have suggested that the gut microbiota also acts as a major modulator of systemic innate immunity during sepsis. Microbiota disruption by broad-spectrum antibiotics could thus have adverse effects on cellular responsiveness towards invading pathogens. As such, the use of antibiotics may attribute to immunosuppression as seen in sepsis. We aimed to test whether disruption of the gut microbiota affects systemic innate immune responses during endotoxemia in healthy subjects. Design In this proof-of-principle intervention trial, 16 healthy young men received either no treatment or broad-spectrum antibiotics (ciprofloxacin, vancomycin and metronidazole) for 7 days, after which all were administered lipopolysaccharide intravenously to induce a transient sepsis-like syndrome. At various time points, blood and faeces were sampled. Results Gut microbiota diversity was significantly lowered by the antibiotic treatment in all subjects. Clinical parameters, neutrophil influx, cytokine production, coagulation activation and endothelial activation during endotoxemia were not different between antibiotic-pretreated and control individuals. Antibiotic treatment had no impact on blood leucocyte responsiveness to various Toll-like receptor ligands and clinically relevant causative agents of sepsis (Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli) during endotoxemia. Conclusions These findings suggest that gut microbiota disruption by broad-spectrum antibiotics does not affect systemic innate immune responses in healthy subjects during endotoxemia in humans, disproving our hypothesis. Further research is needed to test this hypothesis in critically ill patients. These data underline the importance of translating findings in mice to humans.