Initial microbial colonization plays an important role in neonatal gut health. However, studies on gut microbial composition at birth are challenging, due to the limited access to accurate sampling. Here, we characterized the jejunal and ileal bacterial composition (epimural and luminal) of neonatal calves within 30 minutes after birth, and compared it with maternal (birth canal and rectum) and birth environments. RNA-based quantification along with amplicon sequencing revealed the colonization of active, dense (1.1–9.4 × 108 16S rRNA copy/g of sample), and diverse bacteria in the calf small intestine at birth. Pseudomonadaceae and Propionibacteriaceae dominated epimural communities, while Propionibacteriaceae, Prevotellaceae, Ruminococcaceae, and Lachnospiraceae dominated luminal communities. The composition of calf gut bacteria at birth was significantly different from maternal bacteria, especially for beneficial bifidobacteria. The bacterial communities of calf body habitats were similar to those of the birth environment, which was again divergent from gut microbiota. This study suggests an establishment of small intestinal-specific microbiota from birth, which is considerably deviated from maternal microbiota. In corollary, we further propose that small intestinal microbiota colonization could be mainly modulated by host selection.