The alternative sigma factor RpoS in Borrelia burgdorferi plays a central role in modulating host adaptive responses when spirochaetes cycle between ticks and mammals. The transcriptional activation of σ⁵⁴-dependent rpoS requires a Fur homologue designated BosR. Previously, BosR was shown to directly activate rpoS transcription by binding to the rpoS promoter. However, many other DNA binding features of BosR have remained obscure. In particular, the precise DNA sequence targeted by BosR has not yet been completely elucidated. The prediction of a putative Per box within the rpoS promoter region has further confounded the identification of the BosR binding sequence. Herein, by using electrophoretic mobility shift assays, we demonstrate that the putative Per box predicted in the rpoS promoter region is not involved in the binding of BosR. Rather, a 13 bp palindromic sequence (ATTTAANTTAAAT) with dyad symmetry, which we denote as the ‘BosR box’, functions as the core sequence recognized by BosR in the rpoS promoter region of Borrelia burgdorferi. Similar to a Fur box and a Per box, the BosR box probably comprises a 6–1–6 inverted repeat composed of two hexamers (ATTTAA) in a head-to-tail orientation. Selected mutations in the BosR box prevented recombinant BosR from binding to rpoS. In addition, we found that sequences neighbouring the BosR box also are required for the formation of BosR–DNA complexes. Identification of the BosR box advances our understanding of how BosR recognizes its DNA target(s), and provides new insight into the mechanistic details behind the unique regulatory function of BosR.