Genetic manipulation of Antarctic bacteria has been very limited so far. This article reports the isolation and molecular characterization of a novel plasmid, pMtBL, from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAC 125. This genetic element, 4,081 bp long, appeared to be a multicopy cryptic replicon with no detectable transcriptional activity. By an in vivo assay, the pMtBL autonomous replication sequence was functionally limited to an AluI plasmid fragment of about 850 bp. This novel cold-adapted replication element showed quite a broad host range profile: it was cloned into a mesophilic genetic construction, obtaining a cold-adapted expression vector that was able to promote the production of P. haloplanktis A23 alpha-amylase in a psychrophilic bacterium. This study represents the first report of successful recombinant production of a cold-adapted protein in an Antarctic host.