Polyamines decrease the permeability of the outer membrane of Escherichia coli to fluoroquinolones and β-lactams. In this study, we tested the effect of four polyamines (spermidine, spermine, cadaverine and putrescine) on fluoroquinolone uptake in Mycobacterium bovis BCG. Our results show that polyamines are also capable of reducing the permeability of the mycobacterial outer membrane to fluoroquinolones. Spermidine was most effective and demonstrated reversible dose- and pH-dependent inhibition of ciprofloxacin accumulation. The extent of this inhibition was demonstrated across the fluoroquinolone compound class to varying degrees. Furthermore, we have shown that the addition of spermidine increases the survival of M. bovis BCG after a 5-day exposure to ciprofloxacin by up to 25 times. The treatment of actively-replicating Mycobacterium tuberculosis with spermidine reduced ciprofloxacin accumulation by half while non-replicating nutrient-starved M. tuberculosis cultures lacked similar sensitivity to polyamines. Gene expression studies showed that several outer membrane proteins are significantly down-regulated during the shift to non-replication. Collectively, these characteristics of fluoroquinolone uptake in M. bovis BCG are consistent with facilitated transport by porin-like proteins and suggest that a reduction in intracellular uptake contributes to the phenotypic drug resistance demonstrated by M. tuberculosis in the non-replicating state.