The intestinal microbiota are a complex ecosystem influencing the immunoregulation of the human host, providing protection from colonising pathogens and producing SCFA as the main energy source of colonocytes. Our objective was to investigate the effect of dietary fibre exclusion and supplementation on the intestinal microbiota and SCFA concentrations. Faecal samples were obtained from healthy volunteers before and after two 14 d periods of consuming formulated diets devoid or supplemented with fibre (14 g/l). The faecal microbiota were analysed using fluorescent in situ hybridisation and SCFA were measured using GLC. There were large and statistically significant reductions in the numbers of the Faecalibacterium prausnitzii (P ≤ 0·01) and Roseburia spp. (P ≤ 0·01) groups during both the fibre-free and fibre-supplemented diets. Significant and strong positive correlations between the proportion of F. prausnitzii and the proportion of butyrate during both baseline normal diets were found (pre-fibre free r 0·881, P = 0·001; pre-fibre supplemented r 0·844, P = 0·002). A significant correlation was also found between the proportional reduction in F. prausnitzii and the proportional reduction in faecal butyrate during both the fibre-free (r 0·806; P = 0·005) and the fibre-supplemented diet (r 0·749; P = 0·013). These findings may contribute to the understanding of the association between fibre, microbiota and fermentation in health, during enteral nutrition and in disease states such as Crohn's disease.