Onconase (ONC) is currently in Phase III clinical trials as a cancer chemotherapeutic agent. Despite the finding that ONC contains an N-linked glycosylation site (-N69-V70-T71-), only the non-glycosylated form of the protein has been identified to date. We employed the Pichia pastoris expression system to produce recombinant glycosylated ONC (gONC) protein. Approximately 10 mg of ONC protein was secreted per liter of culture media, of which about 80% was glycosylated at N69. CD spectroscopic analyses revealed that the secondary structure of gONC is identical to that of ONC. We found that gONC contains a high-mannose core structure. Importantly, glycosylation of ONC at N69 greatly increased its toxicity for K-562 cancer cells. Specifically, the IC50 value of gONC was 50-fold lower than that of ONC. Glycosylation increased both the Tm of ONC and its resistance to proteinase K, suggesting that the elevated cytotoxicity of gONC is related to higher conformational stability.