α1-Antitrypsin (AAT), a serine protease inhibitor, is the third most abundant protein in plasma. Although the best-known function of AAT is irreversible inhibition of elastase, AAT is an acute-phase reactant and is increasingly recognized to have a panoply of other functions, including as an anti-inflammatory mediator and a host-protective molecule against various pathogens. Although a canonical receptor for AAT has not been identified, AAT can be internalized into the cytoplasm and is known to affect gene regulation. Because AAT has anti-inflammatory properties, we examined whether AAT binds the cytoplasmic glucocorticoid receptor (GR) in human macrophages. We report the finding that AAT binds to GR using several approaches, including coimmunoprecipitation, mass spectrometry, and microscale thermophoresis. We also performed in silico molecular modeling and found that binding between AAT and GR has a plausible stereochemical basis. The significance of this interaction in macrophages is evinced by AAT inhibition of LPS-induced NF-κB activation and IL-8 production as well as AAT induction of angiopoietin-like 4 protein, which are, in part, dependent on GR. Furthermore, this AAT-GR interaction contributes to a host-protective role against mycobacteria in macrophages. In summary, this study identifies a new mechanism for the gene regulation, anti-inflammatory, and host-defense properties of AAT.Copyright © 2022 by The American Association of Immunologists, Inc.