<p>Mobile group II introns are site-specific retrotransposons composed of a large self-splicing ribozyme and an intron-encoded reverse transcriptase that are widespread in bacterial and organellar genomes. Sequence and structural variations of the ribozyme and the associated reverse transcriptase define several lineages of bacterial group II introns. Interestingly, some of these intron families evolved different mobility strategies while others colonize particular genetic contexts. Here, we have investigated the mobility activity of an <i>Escherichia coli</i> group II intron that is inserted into the stop codon of the stress-response gene <i>groEL</i>. Using mobility assays based on over-expression from a donor plasmid, we demonstrate that this intron is a highly efficient and site-specific retrotransposon, capable of colonizing the <i>groEL</i> gene of an <i>E. coli</i> host strain according to the insertion pattern observed in natural genomes. Furthermore, we provide evidence that a chromosomal copy of the full-length retrotransposon can be expressed from its native genetic locus to yield mobile retroelement particles. This intron constitutes a novel model system that could help reveal original mobility strategies used by some group II intron retrotransposons to colonize bacterial genomes.</p>