Glycosylation is fundamentally important to the activity, stability and pharmacokinetics of biopharmaceuticals. Move on to the future, glycoengineering is a promising way to improve protein drug efficacy. Recently, several major advances in engineering efforts have successfully led to desired protein glycoforms, but some desirable glycoengineering designs have proven toxic to the host cells (e.g., CHO cell lines). Up to date, the critical barrier in controlling the glycan structures is the diversity and complexity of glycosylation. Moreover, there has not been a systematic study unraveling how engineering efforts impact the host cell and their protein secretion system. There is a great need to bridge the knowledge gap that enables rational glycoengineering and elucidate how such strategies impact the host cell. To accomplish this, we have comprehensively studied the impact of glycoengineering on a library of more than 180 CHO cell clones, wherein dozens of glycosyltransfersases were knocked out or knocked in. We integrate data that glycoprofiled all these clones, quantified the impact of different glycosyltransferase knockouts on the bioprocessing phenotypes of the CHO cells (e.g., cell size, growth, viability, and metabolism), and expression profiling these cells using RNA‐Seq data to identify dysregulated pathways and genes following glycoengineering. Here, we deployed an innovative systems biology approach to rapidly study the changes in glycosylation across all glycoengineered mutants and to study the molecular basis of the phenotypic changes. Our key results are (1) we identified dominant glycosyltransferses responsible for the N‐linked glycosylation in CHO cells, and the glycoengineered cells differentially expressed isozymes in response to a knockout; (2) we identified groups of glycosyltransferases whose perturbations led to a more severe impact on cell glycosylation and phenotypes; (3) we also identified key metabolic and signaling pathways are modulated when different glycosyltransferase families were perturbed. This research is the first study for gaining a comprehensive view of the complex cellular response to glycoengineering on the host cells producing the recombinant protein drugs. The insights from this study provides valuable knowledge that enables the rational engineering of glycosylation to be able to control this critical quality attribute on diverse recombinant protein drugs.