Genetic engineering of oligosaccharide biosynthesis pathways in mammalian cells makes possible generation of new recombinant glycoproteins of potential importance in the biopharmaceutical industry. Most prior investigations of glycosylation engineering of secreted heterologous glycoproteins involve terminal steps of oligosaccharide biosynthesis. In particular, increasing the frequency of bisected structures within the glycoform distribution has not before been considered. A Chinese hamster ovary (CHO) cell line capable of producing bisected oligosaccharides on glycoproteins was created by overexpression of a recombinant N-acetylglucosaminyltransferase III (GnT-III). Interferon beta (IFN-beta) was chosen as a model and potential therapeutic secreted heterologous protein to demonstrate the effect of recombinant GnT-III-expression on product glycosylation. IFN-beta with bisected oligosaccharides was produced by the GnT-III-engineered CHO cells but not by the unmodified parental cell line.