RpoS and RpoN are two alternative sigma factors typically associated with general stress responses in bacteria. To date, there has been no experimental evidence that RpoS and RpoN can directly control the expression of one another. Herein, using a combined strategy of gene disruption and genetic complementation targeting rpoN and rpoS in Borrelia burgdorferi strain 297, we describe a regulatory network for B. burgdorferi. In this network, RpoN controls the expression of RpoS, which, in turn, governs the expression of two important membrane lipoproteins, outer surface protein C and decorin-binding protein A, and likely other proteins of B. burgdorferi. Our findings provide a foundation for elucidating further key regulatory networks that potentially impact many aspects of B. burgdorferi's parasitic strategy, host range, and virulence expression.