ABC transporters are key determinants of bacterial adaptation, yet their functional plasticity remains poorly understood. Here, we characterize the type I ABC transporter YhdWXYZ and uncover a striking functional divergence linked to the presence of its substrate-binding protein (SBP). In Escherichia coli K-12, where yhdW is a pseudogene, deletion of yhdWXYZ increases susceptibility to mecillinam and lomefloxacin and leads to intracellular accumulation of lomefloxacin, indicating a role in antibiotic homeostasis. In contrast, in Citrobacter rodentium , which encodes a complete YhdWXYZ system, deletion of the transporter does not affect antibiotic susceptibility. Biochemical analyses demonstrate that the YhdW SBP of C. rodentium binds asparagine with high affinity; however, genetic and physiological assays indicate that YhdWXYZ is not a primary asparagine importer under laboratory conditions, suggesting redundancy with other transport systems. Importantly, in vivo infection experiments reveal that YhdWXYZ contributes to early colonization and persistence in the host, as mutants display reduced bacterial loads and altered intestinal pathology in mice. Together, these findings show that loss of the SBP in E. coli is associated with a functional shift of YhdWXYZ toward antibiotic homeostasis, whereas in C. rodentium , the complete transporter contributes to host adaptation. This work highlights the evolutionary and functional flexibility of ABC transporters in bacterial physiology and pathogenesis.