Ergothioneine (EGT) is a thiol-based antioxidant synthesized by certain fungal and bacterial species that is prevalent in the human diet. Recently, an EGT-specific transporter, EgtUV, was discovered in bacteria that are incapable of EGT biosynthesis, including the gastric pathogen Helicobacter pylori. However, EGT is naturally abundant in the complex media required to culture H. pylori and many other host-associated microbes, complicating efforts to understand how this molecule influences microbial physiology. Using the solute-binding domain of H. pylori EgtUV, we generated an EGT-chelating resin that depletes EGT from nutrient-rich media. We determined that wild-type H. pylori requires EGT to outcompete a transporter-deficient strain in vitro. Furthermore, EGT induces transcription of genes encoding outer-membrane transporters that may regulate intracellular EGT content upstream of the inner-membrane-localized EgtUV transporter. Our work establishes a method for tuning exposure to an abundant antioxidant in vitro, enabling future studies of EGT in diverse microbial strains and communities.