The blaNDM-1 gene encodes a carbapenemase that confers resistance to almost all β-lactams, including last-resort carbapenems. This is increasingly reported worldwide in nosocomial and community-acquired Gram-negative bacteria. Acinetobacter baumannii is an important opportunistic pathogen that is considered an intermediate reservoir for the blaNDM-1 gene. In this species, the blaNDM-1 gene is located within the Tn125 composite transposon. The mechanism driving the mobility of Tn125 has not yet been elucidated. Here we experimentally demonstrated the transposition of Tn125 in A. baumannii Systematic 3-bp duplication of the target site, being the signature of transposition, was evidenced. The target site consensus sequence for Tn125 transposition was found to be GC enriched at the duplicated 3 bp and AT rich in the vicinity. Transposition frequency was not influenced by temperature changes or by exposure to subinhibitory concentrations of various antibiotics. This work is the first direct evidence of the functionality of a composite transposon in A. baumannii It provides a mechanistic clue for the dissemination of the blaNDM-1 gene in Acinetobacter spp. and subsequently among Enterobacteriaceae.