Polyamines are an essential class of metabolites found throughout all kingdoms in life. Borrelia burgdorferi harbors no enzymes to synthesize or degrade polyamines yet does contain a polyamine uptake system, potABCD. In this report, we describe the initial characterization of this putative transport system. After several unsuccessful attempts to inactivate potABCD, we placed the operon under the control of an inducible LacI promoter expression system. Analyses of this construct confirmed that potABCD was required for in vitro survival. Additionally, we demonstrated that the potABCD operon were upregulated in vitro by low osmolarity. Previously, we had shown that low osmolarity triggers the activation of the Rrp2/RpoN/RpoS regulatory cascade, which regulates genes essential for the transmission of spirochetes from ticks to mammalian hosts. Interestingly, induction of the pot operon was only affected in an rpoS mutant but not in a rpoN mutant, suggesting that the genes were RpoS dependent and RpoN independent. Furthermore, potABCD was upregulated during tick feeding concomitant with the initiation of spirochete replication. Finally, uptake experiments determined the specificity of B. burgdorferi's PotABCD for spermidine.