The alternative sigma factor RpoS (σ38 or σS) plays a central role in the reciprocal regulation of the virulence-associated major outer surface proteins OspC and OspA in Borrelia burgdorferi, the Lyme disease spirochete. Temperature is one of the key environmental signals controlling RpoS, but the molecular mechanism by which the signal is transduced remains unknown. Herein, we identify and describe a small non-coding RNA, DsrABb, that regulates the temperature-induced increase in RpoS. A novel 5′ end of the rpoS mRNA was identified and DsrABb has the potential to extensively base-pair with the upstream region of this rpoS transcript. We demonstrate that B. burgdorferi strains lacking DsrABb do not upregulate RpoS and OspC in response to an increase in temperature, but do regulate RpoS and OspC in response to changes in pH and cell density. Analyses of the rpoS and ospC steady-state mRNA levels in the dsrABb mutant indicate that DsrABb regulates RpoS post-transcriptionally. The 5′ and 3′ ends of DsrABb were mapped, demonstrating that at least four species exist with sizes ranging from 213 to 352 nucleotides. We hypothesize that DsrABb binds to the upstream region of the rpoS mRNA and stimulates translation by releasing the Shine-Dalgarno sequence and start site from a stable secondary structure. Therefore, we postulate that DsrABb is a molecular thermometer regulating RpoS in Borrelia burgdorferi.