Constipation is one of the most common gastrointestinal complaints worldwide. This study was performed to determine whether Bifidobacterium adolescentis exerts inter-strain differences in alleviating constipation induced by loperamide in BALB/c mice and to analyze the main reasons for these differences. BALB/c mice underwent gavage with B. adolescentis (CCFM 626, 667, and 669) once per day for 17 days. The primary outcome measures included related constipation indicators, and the secondary outcome measures were the basic biological characteristics of the strains, the concentration changes of short-chain fatty acids in feces, and the changes in the fecal flora. B. adolescentis CCFM 669 and 667 relieved constipation symptoms by adhering to intestinal epithelial cells, growing quickly in vitro and increasing the concentrations of propionic and butyric acids. The effect of B. adolescentis on the gut microbiota in mice with constipation was investigated via 16S rRNA metagenomic analysis. The results revealed that the relative abundance of Lactobacillus increased and the amount of Clostridium decreased in the B. adolescentis CCFM 669 and 667 treatment groups. In conclusion, B. adolescentis exhibits strain-specific effects in the alleviation of constipation, mostly due to the strains' growth rates, adhesive capacity and effects on the gut microbiome and microenvironment.