Phytol is a naturally occurring precursor of phytanic acid. The last step in the conversion of phytol to phytanoyl-CoA is the reduction of phytenoyl-CoA mediated by an, as yet, unidentified enzyme. A candidate for this reaction is a previously described peroxisomal trans-2-enoyl-CoA reductase (TER). To investigate this, human TER was expressed in E. coli as an MBP-fusion protein. The purified recombinant protein was shown to have high reductase activity towards trans-phytenoyl-CoA, but not towards the peroxisomal beta-oxidation intermediates C24:1-CoA and pristenoyl-CoA. In conclusion, our results show that human TER is responsible for the reduction of phytenoyl-CoA to phytanoyl-CoA in peroxisomes.