The aim of this study was to explore the effect of colostrum feeding time on the ileal microbiome of neonatal calves. In this study, 22 male Holstein calves were randomly assigned to different colostrum feeding time treatments: after birth (at 45 min, n = 7); at 6 h after birth (n = 8); and at 12 h after birth (TRT12h; n = 7). At 51 h after birth, calves were killed and ileum digesta was collected for microbiome analysis using shotgun metagenomic sequencing. Bacteria, archaea, eukaryotes, and viruses were identified from the ileum microbiome. For the bacteriome, Firmicutes and Proteobacteria were the predominant phyla, and Escherichia, Streptococcus, Lactobacillus were the 3 most abundant genera. For the archaeal community, Euryarchaeota and Crenarchaeota were the 2 major phyla, and Methanosarcina, Methanobrevibacter, and Methanocorpusculum were the 3 most abundant genera. In total, 116 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified from the ileal microbiome, with “biosynthesis of vancomycin group antibiotics,” “biosynthesis of ansamycins,” “valine, leucine, and isoleucine biosynthesis,” “ribosome,” and “D-alanine metabolism” as the top 5 functions. When the ileal microbiomes were compared among the 3 treatments, the relative abundance of Enterococcus was higher in TRT12h calves, suggesting that calves may have a higher abundance of opportunistic pathogens when the feeding of colostrum is delayed for 12 h. Moreover, among all KEGG pathways, the enriched “taurine and hypotaurine metabolism” (KO00430) pathway was identified in the ileal microbiome of TRT12h calves; however, future studies are needed to understand the effect on the host. Additionally, 2 distinct ileal microbial profiles were identified across all samples, indicating that that host factors may play a significant role in driving varied microbiome changes in response to colostrum feeding time. Whether such microbiome shifts affect long-term gut function and calf performance warrants future studies.