Vitamin D (VitD) is emerging as an immune regulator in addition to its established role in metabolism and mineral homeostasis. This study sought to determine if in vivo VitD modulated the oral and faecal microbiome in Holstein–Friesian dairy calves. The experimental model consisted of two control groups (Ctl-In, Ctl-Out) which were fed with a diet containing 6000 IU/Kg of VitD3in milk replacer and 2000 IU/Kg in feed, and two treatment groups (VitD-In, VitD-Out) with 10,000 IU/Kg of VitD3in milk replacer and 4000 IU/Kg in feed. One control and one treatment group were moved outdoors post-weaning at approximately 10 weeks of age. Saliva and faecal samples were collected after 7 months of supplementation and analysis of the microbiome was performed using 16S rRNA sequencing. Bray–Curtis dissimilarity analysis identified that both sampling site (oral vs. faecal) and housing (indoor vs. outdoor) had significant influences on the composition of the microbiome. The calves housed outdoors had greater microbial diversity in the faecal samples based on Observed, Chao1, Shannon, Simpson and Fisher measures in comparison to calves housed indoors (P< 0.05). A significant interaction between housing and treatment was observed for the generaOscillospira, Ruminococcus,CF231andPaludibacterin faecal samples. The generaOscillospiraandDoreawere increased whileClostridiumandBlautiawere decreased following VitD supplementation in the faecal samples (P< 0.05). An interaction between VitD supplementation and housing was detected in the abundance of the generaActinobacillusandStreptococcusin the oral samples. VitD supplementation increased the generaOscillospira,Helcococcusand reduced the generaActinobacillus,Ruminococcus,Moraxella,Clostridium,Prevotella,SuccinivibrioandParvimonas. These preliminary data suggest that VitD supplementation alters both the oral and faecal microbiome. Further research will now be conducted to establish the significance of microbial alterations for animal health and performance.