We reported in an earlier study that active efflux of drug has a predominant role in conferring resistance in a laboratory-generated ciprofloxacin-resistant mutant of Mycobacterium smegmatis. This mutant exhibited mRNA level overexpression, as well as chromosomal amplification, of the gene pstB, encoding the putative ATPase subunit of phosphate specific transport (Pst) system. We demonstrate here that this mutant shows enhanced phosphate uptake and that inactivation of pstB in the parental strain results in loss of high affinity phosphate uptake and hypersensitivity to fluoroquinolones. These findings suggest a novel role of the Pst system in active efflux, in addition to its involvement in phosphate transport.