Ruminants thrive in diverse ecosystems by leveraging their rumen microbiome to ferment fibrous plants. However, the spatial biogeography of rumen microbiome and the genetic diversity of the ventral rumen epithelium remain unknown. Here, we present a multi-omics study in roe deer, sika deer, and sheep, integrating region-resolved microbiome and metabolome across 11 ruminal sacs, as well as single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), and bulk RNA sequencing (RNA-seq) of ventral epithelium. We reveal species-specific microbial compositions and metabolic capacities contributing to differences in short-chain fatty acid and vitamin B production. We uncover functional divergence, genomic specialization, and metabolic changes across the microbiome of distinct ruminal sacs. Single-cell profiling reveals changes of immune responses and structural remodeling of the ruminal ventral epithelium. We demonstrate that vitamin B12 promotes epithelial growth and we identify genes enhancing stem cell differentiation. Our results highlight variation in microbial ecology and epithelial architecture among three ruminant species, offering insights to improve livestock productivity.