The cytosolic peptide:N-glycanase (PNGase) is involved in the quality control of N-glycoproteins via the endoplasmic reticulum-associated degradation (ERAD) pathway. Mutations in the gene encoding cytosolic PNGase (NGLY1 in humans) cause NGLY1 deficiency. Recent findings indicate that the F-box protein FBS2 of the SCFFBS2 ubiquitin ligase complex can be a promising drug target for NGLY1 deficiency. Here, we determined the crystal structure of bovine FBS2 complexed with the adaptor protein SKP1 and a sugar ligand, Man3GlcNAc2, which corresponds to the core pentasaccharide of N-glycan. Our crystallographic data together with NMR data revealed the structural basis of disparate sugar-binding specificities in homologous FBS proteins and identified a potential druggable pocket for in silico docking studies. Our results provide a potential basis for the development of selective inhibitors against FBS2 in NGLY1 deficiency.