CHO glycosylation mutants, pioneered by Stanley and co-workers, have proven to be valuable tools in glycobiology and biopharmaceutical research. Here we aim to provide a summary of our efforts to isolate industrially applicable CHO glycosylation mutants, termed CHO-gmt cells, using cytotoxic lectins and zinc-finger nuclease technology. The genetic defects in the glycosylation machinery in these cells lead to the production of recombinant glycoproteins with consistent and unique glycan structures. In addition, these mutant cells can be easily adapted to serum-free medium in suspension cultures, the condition used by the biotech industry for large-scale production of recombinant therapeutics. In light of the critical impact of glycosylation on biopharmaceutical performances, namely, safety and efficacy, the CHO-gmt lines have enormous potential in producing glycoprotein therapeutics with optimal glycosylation profiles, thus, representing a panel of ideal host cell lines for producing recombinant biopharmaceuticals with improved safety profiles and enhanced efficacy.