Canonical mechanisms of antimicrobial resistance (AMR) enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. However existing AMR genes do not explain all antibiotic-resistant infections. We have defined an entirely new mechanism of resistance to the antibiotic sulfamethoxazole that is distinct from described mechanisms of resistance and not annotated in AMR gene databases. Resistance is mediated by a protein (ThfT) that modifies the substrate profile of an endogenous ABC transporter to include the end products of the metabolic pathway inhibited by sulfamethoxazole. As this mechanism of AMR involves acquisition of these metabolites (tetrahydrofolate and related compounds) directly from the host, it remains undetectable by in vitro antibiotic susceptibility testing methods. We propose that ThfT-mediated AMR constitutes a new paradigm of bacterial AMR that is only active during an infection. As ThfT-mediated resistance is undetectable by routine surveillance methods, our study highlights the need to understand antibiotic susceptibility during infection to reduce inappropriate antibiotic use and treatment failures.