Tigecycline resistance remains rare amongst Enterobacteriaceae in the UK, as elsewhere, but has been associated with upregulation of the AcrAB efflux system. Using isolates of an Enterobacter cloacae strain that developed tigecycline resistance in vivo during ciprofloxacin therapy as well as laboratory-selected mutants, we investigated the role of this pump and the global regulator RamA in tigecycline resistance. Laboratory mutants were selected from a susceptible clinical isolate in vitro by exposure to increasing concentrations of tigecycline. Expression of the acrAB operon and the ramA gene was monitored by real-time reverse-transcription polymerase chain reaction (RT-PCR). Overexpression of ramA was achieved using the pBAD expression vector, whilst insertional inactivation of acrB with a gentamicin resistance cassette was achieved with the bacteriophage lambda Red recombination system. Increased tigecycline minimum inhibitory concentrations in the clinical isolate and a laboratory mutant were associated with increases in acrAB and ramA transcripts. Induction of increased ramA expression resulted in increased acrAB expression, whilst insertional inactivation of acrB restored full susceptibility to tigecycline. Treatment with ciprofloxacin, a substrate of AcrAB in E. cloacae, possibly selected for cross-resistance to tigecycline as a result of RamA-mediated AcrAB upregulation.