The bioactivity of lignans depends on their transformation by gut bacteria. The intestinal bacteria Clostridium saccharogumia, Eggerthella lenta, Blautia producta and Lactonifactor longoviformis convert the plant lignan secoisolariciresinol diglucoside via secoisolariciresinol (SECO) into the bioactive enterolignans enterodiol (ED) and enterolactone (EL). While the in vitro conversion of lignans by these bacteria has already been demonstrated, it is unclear whether this defined community is also capable of catalysing lignan transformation in vivo. We therefore associated germ-free rats with these four species. Germ-free rats served as a control. All animals were fed a diet containing 5% ground flaxseed. The caecal contents of rats associated with the four lignan-activating bacteria (ALB rats) contained SECO, ED and EL. The maximal EL formation rate from lignans in the pooled caecal contents of ALB rats was 7.52 nmol min⁻¹ g⁻¹ dry matter. The ALB rats excreted EL, but no SECO and ED, in their urine. The caecal contents of germ-free rats contained SECO, but no ED and EL. Their urine was devoid of lignans. Hence, the presence of enterolignans in the ALB rats, but not in the germ-free rats, demonstrates that this defined microbial community is capable of transforming plant lignans into EL in vivo.