Enrichment of calf diets with exogenous butyrate has shown promise as a promotor of calf growth and intestinal development. However, the impact of dietary derived butyrate on the gut microbiota and their potential role, in turn, as mediators of its effect on calf growth and development is not known. Here, the effects of butyrate supplementation on rumen and hindgut microbiota and fermentation profiles were assessed in 16 Holstein-Friesian bull calves randomly assigned to one of two groups: Control (CON) fed conventional milk replacer or Sodium-Butyrate (SB - added to milk replacer) from days 7 to 56 of life. In the colon, total short chain fatty acid (SCFA), propionate and acetate concentrations were increased by SB (P < 0.05). 16S rRNA gene amplicon sequencing showed cecal abundance of butyrate producers Butyrivibrio and Shuttleworthia were decreased by SB (P < 0.05), while that of the propionate producer Phascolarctobacterium was higher (P < 0.05). Mogibacterium is associated with impaired gut health and was reduced in the cecum of SB calves (P < 0.05). These data show that the beneficial effects of SB on growth and performance occur in tandem with changes in the abundance of important SCFA producing and health-associated bacteria in the hindgut in milk-fed calves.