We describe a small-molecule ‘walker’ that uses enzyme catalysis to discriminate between the relative positions of its ‘feet’ on a track and thereby move with net directionality. The bipedal walker has identical carboxylic acid feet, and ‘steps’ along an isotactic hydroxyl-group-derivatized polyether track by the formation/breakage of ester linkages. Lipase AS catalyzes the selective hydrolysis of the rear foot of macrocyclized walkers (an information ratchet mechanism), the rear foot producing an (R)-stereocenter at its point of attachment to the track. If the hydrolyzed foot re-attaches to the track in front of the bound foot it forms an (S)-stereocenter, which is resistant to enzymatic hydrolysis. Only macrocyclic walker-track conjugates are efficiently hydrolyzed by the enzyme, lead-ing to high processivity of the walker movement along the track. Conventional chemical reagents promote formation of the ester bonds between the walker and the track. Iter-ative macrocyclization and hydrolysis reactions lead to 68 % of walkers taking two steps directionally along a three-foothold track.