Peptide:N-glycanase (PNGase) is an enzyme responsible for deglycosylation of misfolded glycoproteins in so-called endoplasmic reticulum-associated degradation (ERAD) system. In this study, we reported the molecular identification and characterization of SpPNGase (Schizosaccharomyces pombe PNGase). Enzymatic analysis revealed that SpPNGase deglycosylated the misfolded glycoproteins and distinguished native and denatured high-mannose glycoproteins in vitro. The deglycosylation activity was lost with the addition of chelating agent EDTA and was not restored by re-addition of metal ions. By construction of deletion mutant, we confirmed that N-terminal alpha-helix of SpPNGase was responsible for the protein-protein interaction. Combining the results from ternary structure prediction and dendrogram analysis, we suggested that the N-terminal alpha-helices of PNGase are derived from evolutionary motif/peptide fusion.