Aberdeen Angus calves were sacrificed from immediately post-birth up to 96 days of age (DOA) and ileal samples were collected for microbial, histological and immunological analyses. Firmicutes bacteria were established immediately in the ileum of calves after birth and remained the dominant phyla at all time points from birth until 96 DOA. Temporal shifts in phyla reflected significantly increased Bacteroidetes at birth followed by temporal increases in Actinobacteria abundance over time. At a cellular level, a significant increase in cell density was detected in the ileal villi over time. The innate cell compartment at birth was composed primarily of eosinophils and macrophages with a low proportion of adaptive T lymphocytes; whereas an increase in the relative abundance of T cells (including those in the intra-epithelial layer) was observed over time. The ileal intestinal cells were immunologically competent as assessed by expression levels of genes encoding the inflammasome sensor NLRP3, and inflammatory cytokines IL1A, IL1B and IL33-all of which significantly increased from birth. In contrast, a temporal reduction in genes encoding anti-inflammatory cytokine IL10 was detected from birth. This study provides an integrated baseline of microbiological, histological and immunological data on the immune adaptation of the neonatal ileum to microbial colonisation in calves.