Cytoplasmic peptide:N-glycanase (PNGase) is an enzyme that removes N-glycans from misfolded glycoproteins. The function of cytoplasmic PNGase plays a significant role in the degradation of misfolded glycoproteins, which is critical for cell viability. Recently, we reported that haloacetoamidyl derivatives of high-mannose-type oligosaccharides selectively modify the catalytic cysteine of cytoplasmic PNGase and serve as its specific inhibitor. Interestingly, a drastically simplified chloroacetamidyl chitobiose derivative [(GlcNAc)(2)-ClAc] was also reactive to PNGase. In our work, it was conjugated to a hydrophobic fluorophore in order to render (GlcNAc)(2)-ClAc cells permeable. We demonstrated that this compound [BODIPY-(GlcNAc)(2)-ClAc] specifically binds to cytoplasmic PNGase from budding yeast (Png1). To date, only Z-VAD-fmk is known as an inhibitor of PNGase. BODIPY-(GlcNAc)(2)-ClAc and Z-VAD-fmk share the same binding site on Png1, while BODIPY-(GlcNAc)(2)-ClAc has markedly stronger inhibitory activity. The functional analysis of PNGase using Z-VAD-fmk should be carefully interpreted because of its intrinsic property as a caspase inhibitor. In sharp contrast, chloroacetamidyl chitobiose was not reactive to caspase. In addition, BODIPY-(GlcNAc)(2)-ClAc did not bind either chitobiose-binding lectins or PNGase from other sources. Moreover, fluorescent microscopy clearly showed that BODIPY-(GlcNAc)(2)-ClAc was efficiently introduced into cells. These results suggest that this compound could be an in vivo inhibitor of cytoplasmic PNGase.