AbstractTwo structurally‐related members of the lysosomal mannosidase family, the broad substrate specificity enzyme human lysosomal α‐mannosidase (hLM, MAN2B1) and the human core α‐1, 6‐specific mannosidase (hEpman, MAN2B2) act in a complementary fashion on different glycosidic linkages, to effect glycan degradation in the lysosome. We have successfully expressed these enzymes in Drosophila S2 cells and functionally characterized them. hLM and hEpman were significantly inhibited by the class II α‐mannosidase inhibitors, swainsonine and mannostatin A. We show that three pyrrolidine‐based compounds designed for selective inhibition of Golgi α‐mannosidase II (GMII) exhibited varying degrees of inhibition for hLM and hEpman. While these compounds inhibited hLM and GMII similarly, they inhibited hEpman to a lesser extent. Further, the two lysosomal α‐mannosidases also show differential metal dependency properties. This has led us to propose a secondary metal binding site in hEpman. These results set the stage for the development of selective inhibitors to members of the GH38 family, and, henceforth, the further investigation of their physiological roles.