It is largely unknown whether the gut microbiome regulates immune responses in humans. We determined relationships between the microbiota composition and immunological phenotypes in 108 healthy volunteers, using 16S sequencing, an ex vivo monocyte challenge model, and an in vivo challenge model of systemic inflammation induced by lipopolysaccharide (LPS).Significant associations were observed between the microbiota composition and ex vivo monocytic cytokine responses induced by several stimuli, most notably IL-10 production induced by Pam3Cys, P. aeruginosa and C. albicans, although the explained variance was rather low (0.3 to 4.8%). Furthermore, a number of pairwise correlations between Blautia, Bacteroides and Prevotella genera and cytokine production induced by these stimuli were identified. LPS administration induced a profound transient in vivo inflammatory response. A second LPS challenge one week after the first resulted in a severely blunted response, reflecting endotoxin tolerance. However, no significant relationships between microbiota composition and in vivo parameters of inflammation or tolerance were found (explained variance ranging from 0.4 to 1.5%, ns).The gut microbiota composition explains a limited degree of variance in ex vivo monocytic cytokine responses to several pathogenic stimuli, but no relationships with the LPS-induced in vivo immune response or tolerance was observed.