Bifidobacteria are members of the human gut microbiota, which are known to influence the metabolic abilities of their host. Here, we investigated the capabilities of bifidobacteria to reduce cholesterol levels in synthetic growth media, clearly demonstrating assimilation of this molecule by particular bifidobacterial strains, including Bifidobacterium bifidum PRL2010 (LMG S-28692). The transcriptomic analysis of PRL2010 cells cultivated in the presence of cholesterol revealed a significantly increased transcription level of genes encoding putative transporters and reductases, indicative of specific mechanisms for cholesterol assimilation as well as cholesterol conversion to coprostanol. Cholesterol lowering activity of B. bifidum PRL2010 cells was further evaluated by means of an in vivo murine model, showing that the fecal microbiota of mice is modified toward those bacteria involved in the metabolism of cholesterol. © 2015, Springer-Verlag Berlin Heidelberg. ; We thank GenProbio srl for financial support of the Laboratory of Probiogenomics. This work was financially supported by a FEMS Jensen Award to FT. FT is a members of The Alimentary Pharmabiotic Centre, while DvS is also a member of the Alimentary Glycoscience Research Cluster, both funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (Grant numbers SFI/12/RC/2273 and 08/SRC/B1393, respectively). ; Peer Reviewed