Microbial colonization can influence the early development and health of dairy calves. To better understand early life bacterial colonization in the calf gastrointestinal tract (GIT), this study investigated (1) the shift in bacterial groups present at the colon mucosa between birth and 5 d old, and (2) the relationship between short chain fatty acid (SCFA) and bacterial composition at 7 d of age in GIT digesta. Holstein bull calves (n = 20) were natural delivered, directly removed from the dam, and housed in individual straw-bedded pens. Calves received 2 meals of a standardized colostrum at 2 h (7% of BW; 180g of IgG fed) and 12 h (3% of BW; 120g of IgG fed) postnatal. Milk replacer (7.5% of BW; 260 g/kg CP; 160 g/kg crude fat, at 150g/L) was bottle-fed twice daily. Colonic endoscopic biopsies were performed within 2 h and at 5 d postnatal. Calves were euthanized at 7 d of age to obtain jejunal, ileal and colonic digesta. Data from colon biopsies were compared between d0 and d5 and data collected at d7 were compared among digesta (jejunum, ileum, and colon). Calf was used as random effect in the models. Real-time quantitative PCR (qPCR) revealed that the total bacterial density in the colon-mucosa decreased between birth and 5 d old (respectively, 9.18 ± 0.16 vs 8.57 ± 0.07 log₁₀ 16S rRNA genes copy number/g, P < 0.01). The relative abundances of Bifidobacterium spp., Lactobacillus spp.,and F. prausnitzii were increased from birth to d 5 (P < 0.01), whereas E. coli significantly decreased (P < 0.01) at d 5 (0.37 ± 0.09% of total bacteria) compared with birth (10.60 ± 1.90% of total bacteria). Total bacterial density was higher (P < 0.01) in colon compared with the ileum and jejunum (colon: 9.37 ± 0.07; ileum: 8.81 ± 0.11; jejunum: 8.12 ± 0.10 log₁₀/g). Total SCFA concentration was higher (P = 0.03) in the colon (12.73 ± 1.12 µmol/mL) compared with the ileum (6.68 ± 0.40 µmol/mL) and jejunum (4.92 ± 0.30 µmol/mL). Acetate was the predominant SCFA throughout the GIT (>90% of total SCFA). In calves, there is an early shift of bacterial colonization in the colon-mucosa and major fermentation activity in the colon with a high density of bacteria in digesta.