This study aimed to characterize mucosa- and digesta-associated microbiota in the hindgut (cecum, colon and rectum) of newborn (NB, n = 6), day 7 (n = 6), day 21 (n = 6) and day 42 (n = 6) Holstein bull calves using amplicon sequencing. The hindgut microbiota was diverse at birth, and mucosa-attached microbial community had higher individual variation than that of digesta-associated community. In total, 16 phyla were identified with Firmicutes, Bacteroidetes and Proteobacteria being the dominant microbial taxa in the hindgut. Quantitative real-time PCR analysis showed a significant age effect on the proportion of mucosa-attached Escherichia coli, Bifidobacterium, Clostridium cluster XIVa and Faecalibacterium prausnitzii. Especially, high abundance of mucosa-associated Escherichia was detected during the first week of life, suggesting higher chance of the pathogenic infection during this stage. The relative abundances of predicted microbial genes involved in amino acid metabolism, carbohydrate metabolism and energy metabolism were enriched, indicating the importance of hindgut microbiota in fermentation during the pre-weaned period. Moreover, the significant correlation between short-chain fatty acid concentration and mucosa-attached carbohydrate utilizing (Coprococcus 1, Blautia, Lachnospiraceae NC2004 group, etc.) and health-related bacteria (Escherichia-Shigella and Salmonella) suggests the importance of hindgut microbiota in the fermentation and health of dairy calves during pre-weaned period.