Alpha-1-antitrypsin (A1AT) is a multifunctional, clinically important, high-value therapeutic glycoprotein that can be used for the treatment of many diseases, such as A1AT deficiency, diabetes, graft-versus-host disease, cystic fibrosis, and various viral infections. Currently, the only U.S. food and drug administration-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT (pdA1AT). In addition to its limited supply, this approach poses a risk of infection transmission, since it uses therapeutic A1AT harvested from donors. To address these issues, we sought to generate recombinant human A1AT (rhA1AT) that is comparable to its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By perturbing nine key genes that are part of the CHO glycosylation machinery and expressing the human ST6GAL1 and A1AT genes, we obtained stable, high producing geCHO-L lines that produced rhA1AT having a highly similar glycoprofile to pdA1AT. Additionally, the rhA1AT demonstrated in vitro activity and in vivo half-life comparable to commercial pdA1AT. Thus, we anticipate that this platform will help produce human-like recombinant plasma proteins, thereby providing a more sustainable and reliable source of therapeutics that are cost-effective and better-controlled regarding purity, clinical safety, and quality.