BosR, the sole member of the ferric uptake regulator (FUR) family in Borrelia burgdorferi ( Bb), is essential for the spirochete’s transcriptional adaptation to the mammalian host environment. Although best known for activating rpoS and establishing the mammalian-phase RpoS regulon, BosR originally was linked to regulation of genes involved in Bb’s oxidative stress response. Here, we show that BosR governs gene expression through both RpoS-dependent and RpoS-independent mechanisms under in vitro and mammalian host-adapted conditions. Using RNA-seq and a DNA-binding-defective BosR-R39A mutant, we demonstrate that DNA binding is essential for BosR’s global regulatory functions. BosR activates rpoS, promotes RpoS-dependent gene regulation, and independently modulates a distinct set of genes involved in a variety of cellular functions, including genome maintenance, chemotaxis, and virulence. Notably, canonical oxidative stress response genes previously attributed to BosR were not differentially expressed in Δ bosR strains in vitro or in mammals. Despite its broad regulatory scope, BosR does not recognize a single, conserved DNA-binding motif, suggesting that DNA occupancy is influenced by local sequence context or DNA topology. Our findings support a bifunctional model in which BosR collaborates with RNA polymerase (RNAP)-RpoS holoenzyme to activate and repress RpoS-regulated genes, while functioning as a canonical FUR-like regulator to control RpoD-dependent genes independently of RNAP interaction.