The small-molecular compound miglustat (N-butyldeoxynojirimycin, Zavesca(®)) has been approved for clinical use in type 1 Gaucher disease and Niemann-Pick type C disease, which are disorders caused by dysfunction of the endosomal-autophagic-lysosomal system. Miglustat inhibits a number of enzymes involved in glycoconjugate and glycan metabolism, including β-glucosidase 2 (GBA2), which is exceptionally sensitive to inhibition by miglustat. GBA2 is a glucosylceramide-degrading enzyme that is located on the plasma membrane/endoplasmic reticulum, and is distinct from the lysosomal enzyme glucocerebrosidase (GBA). Various strands of evidence suggest that inhibition of GBA2 contributes to the therapeutic benefits of miglustat. To further explore the pharmacology and biology of GBA2, we investigated whether the zebrafish homolog of GBA2 has similar enzymatic properties and pharmacological sensitivities to its human counterpart. We established that zebrafish has endogenous β-glucosidase activity toward lipid- and water-soluble GBA2 substrates, which can be inhibited by miglustat, N-butyldeoxygalactonojirimycin, and conduritol B epoxide. β-Glucosidase activities with highly similar characteristics were expressed in cells transfected with the zebrafish gba2 cDNA and in cells transfected with the human GBA2 cDNA. These results provide a foundation for the use of zebrafish in screening GBA2-targeting molecules, and for wider studies investigating GBA2 biology.