The understanding on pre-ruminant gut microbiome is very limited. Therefore, this study characterized the small intestinal (jejunum and ileum) microbiome of pre-weaned calves, aiming to understand the dynamics of postnatal microbial establishment within the first 6 weeks of life and how potentially it interacts with host mucosal immune system. Next generation sequencing of digesta- and tissue-associated communities revealed remarkable variations in the microbial composition and the relative abundance of detected bacterial groups among individuals. Despite the highly individualized microbiome, we identified 2 taxonomic-based clusters of calves that were comprised of either high levels of Lactobacillus or Bacteroides. Moreover, Bacteroides, Prevotella, Roseburia, Ruminococcus, and Veillonella were low abundant or absent in the ileum of Lactobacillus-dominant calves. The analysis of metagenome profiles revealed that calves can be grouped to 2 function-based clusters with either high protein metabolism (cluster1) or sulfur metabolism (cluster2). When the small intestinal transcriptome was profiled, it indicates that the first week after birth is a very dynamic developmental period for the intestinal mucosal immune system. Similar changes were observed in the expression of miRNAs and microbiome during the first week of life suggesting that the changes observed at transcriptome level may be regulated by both miRNAs and microbial colonization. Besides, the ileal transcriptome of the calves belonged to 2 taxonomic-based clusters revealed varied immune responses. The present study revealed that an establishment of small intestinal-specific microbiota from birth, and there are microbial markers (microbial functions and taxonomy) that can be used to broadly categorize calves, regardless of the highly individualized early microbiome. Findings from this study indicate that the colonizing microbiome is an essential factor regulating the rapid development of the mucosal immune system during the first week of life.