O-linked N-acetylglucosamine (O-GlcNAc) modification of serines/threonines on cytoplasmic proteins is a significant signal regulating cellular processes such as cell cycle, cell development, and cell apoptosis. O-GlcNAcase (OGA) is responsible for the removal of O-GlcNAc, and it thus plays a critical role in O-GlcNAc metabolism. Interestingly, OGA can be cleaved by caspase-3 into two fragments during apoptosis, producing an N-terminal fragment (1-413 a.a.), termed nOGA. Here, using 4-MU-GlcNAc (4-methylumbelliferyl 2-acetamido-2-deoxy-β-D-glucopyranoside) as substrate, we found that the nOGA fragment retains high glycosidase activity. To probe the role of nOGA in apoptosis, it is essential to develop a potent and specific nOGA inhibitor. However, many reported inhibitors active at nanomolar concentrations (including PUGNAc, STZ, GlcNAc-statin, and NAG-thiazoline) against full-length OGA were not potent for nOGA. Next, we screened a small triazole-linked carbohydrate library and first identified compound 4 (4-pyridyl-1-(2'-deoxy-2'-acetamido-β-D-glucopyranosyl)-1,2,3-triazole) as a potent and competitive inhibitor for nOGA. This compound shows 15-fold selectivity for nOGA (K(i) = 48 μM) over the full-length OGA (K(i) = 725 μM) and 10-fold selectivity over human lysosomal β-hexosaminidase A&B (Hex A&B) (K(i) = 502 μM). These results reveal that compound 4 can be used as a potent and selective inhibitor for probing the role of nOGA in biological systems.