The feeding of heat-treated colostrum is a common practice to increase passive transfer of immunity and decrease enteric infections in calves. However, its influence on gut microbial colonization has not been well studied. The present study investigated the effect of heat-treated colostrum feeding on the bacterial colonization in calf small intestine, within the first 12 h of life. Newborn Holstein bull calves (n = 32) were removed from dams and fed with either fresh colostrum (FC, n = 12) or heat-treated colostrum (HC, n = 12) soon after birth, while the control (NC, n = 8) group did not receive colostrum. Small intestinal samples (tissue and contents) were collected from proximal jejunum, distal jejunum and ileum at 6 and 12 h after birth, following euthanasia. Quantitative real time PCR was used to explore the colonization of total bacteria, lactobacilli, and bifidobacteria. The feeding of colostrum soon after birth increased total bacteria density (FC-1.4 ± 0.7 × 10¹⁰ 16S rRNA copy/g of sample, HC-9.8 ± 3.4 × 10⁹ 16S rRNA copy/g of sample) in calf gut within the first 12 h compared with NC (5.3 ± 2.2 × 10⁸ 16S rRNA copy/g of sample). Although there were bacteria in FC (1.8 × 10³ 16S rRNA gene copy/ml), bacteria were not detectable in HC. In contrast, the prevalence of lactobacilli was lower in HC (0.007 ± 0.004%) and FC (0.014 ± 0.008%), comparing to NC (0.031 ± 0.015%). Remarkable changes in the prevalence of small intestinal tissue-attached bifidobacteria were observed with the feeding of HC soon after birth, but not that in gut contents. The prevalence of bifidobacteria was 3.2-fold and 5.2-fold higher in HC (28.6 ± 10.3%) than FC (8.9 ± 2.5%) and NC (5.5 ± 1.5%), respectively, at 6 h. The feeding of FC increased the prevalence of tissue-attached bifidobacteria gradually, reaching to that of HC at 12 h. Thus, the present study suggests that feeding of HC enhances the colonization of bifidobacteria on small intestinal tissue immediately postpartum compared with that of FC and NC, which may prevent the colonization of enteric pathogens, and thereby decrease enteric infections in neonatal calves.