The vitamin D receptor is highly expressed in the gastrointestinal tract where it transacts gene expression. With current limited understanding of the interactions between the gut microbiome and vitamin D, we conduct a cross-sectional analysis of 567 older men quantifying serum vitamin D metabolites using LC-MSMS and defining stool sub-Operational Taxonomic Units from16S ribosomal RNA gene sequencing data. Faith's Phylogenetic Diversity and non-redundant covariate analyses reveal that the serum 1,25(OH) D level explains 5% of variance in α-diversity. In β-diversity analyses using unweighted UniFrac, 1,25(OH) D is the strongest factor assessed, explaining 2% of variance. Random forest analyses identify 12 taxa, 11 in the phylum Firmicutes, eight of which are positively associated with either 1,25(OH) D and/or the hormone-to-prohormone 1,25(OH) D/25(OH)D "activation ratio." Men with higher levels of 1,25(OH) D and higher activation ratios, but not 25(OH)D itself, are more likely to possess butyrate producing bacteria that are associated with better gut microbial health.