<jats:p>The extract of bark of <jats:italic>Angylocalyx pynaertii</jats:italic> (Leguminosae) was found to potently inhibit mammalian α‐ <jats:sc>l</jats:sc>‐fucosidases. A thorough examination of the extract resulted in the discovery of 15 polyhydroxylated alkaloids, including the known alkaloids from seeds of this plant, 1,4‐dideoxy‐1,4‐imino‐ <jats:sc>d</jats:sc>‐arabinitol (DAB), 1‐deoxymannojirimycin (DMJ) and 2,5‐imino‐1,2,5‐trideoxy‐ <jats:sc>d</jats:sc>‐mannitol (6‐deoxy‐DMDP). Among them, eight sugar‐mimic alkaloids showed the potent inhibitory activity towards bovine epididymis α‐ <jats:sc>l</jats:sc>‐fucosidase and their <jats:italic>K</jats:italic><jats:sub>i</jats:sub> values are as follows: 6‐deoxy‐DMDP (83 µ<jats:sc>m</jats:sc>), 2,5‐imino‐1,2,5‐trideoxy‐ <jats:sc>l</jats:sc>‐glucitol (0.49 µ<jats:sc>m</jats:sc>), 2,5‐dideoxy‐2,5‐imino‐ <jats:sc>d</jats:sc>‐fucitol (17 µ<jats:sc>m</jats:sc>), 2,5‐imino‐1,2,5‐trideoxy‐ <jats:sc>d</jats:sc>‐altritol (3.7 µ<jats:sc>m</jats:sc>), DMJ (4.7 µ<jats:sc>m</jats:sc>), <jats:italic>N</jats:italic>‐methyl‐DMJ (30 µ<jats:sc>m</jats:sc>), 6‐<jats:italic>O</jats:italic>‐α‐ <jats:sc>l</jats:sc>‐rhamnopyranosyl‐DMJ (Rha‐DMJ, 0.06 µ<jats:sc>m</jats:sc>), and β‐ <jats:sc>l</jats:sc>‐homofuconojirimycin (β‐HFJ, 0.0053 µ<jats:sc>m</jats:sc>). We definitively deduced the structural requirements of inhibitors of α‐ <jats:sc>l</jats:sc>‐fucosidase for the piperidine alkaloids (DMJ derivatives). The minimum structural feature of α‐ <jats:sc>l</jats:sc>‐fucosidase inhibitors is the correct configuration of the three hydroxyl groups on the piperidine ring corresponding to C2, C3 and C4 of <jats:sc>l</jats:sc>‐fucose. Furthermore, the addition of a methyl group in the correct configuration to the ring carbon atom corresponding to C5 of <jats:sc>l</jats:sc>‐fucose generates extremely powerful inhibition of α‐ <jats:sc>l</jats:sc>‐fucosidase. The replacement of the methyl group of β‐HFJ by a hydroxymethyl group reduced its inhibitory potential about 80‐fold. This suggests that there may be a hydrophobic region in or around the active site. The existence or configuration of a substituent group on the ring carbon atom corresponding to the anomeric position of <jats:sc>l</jats:sc>‐fucose does not appear to be important for the inhibition. Interestingly, Rha‐DMJ was a 70‐fold more potent inhibitor of α‐ <jats:sc>l</jats:sc>‐fucosidase than DMJ. This implies that the lysosomal α‐ <jats:sc>l</jats:sc>‐fucosidase may have subsites recognizing oligosaccharyl structures in natural substrates.