Insertion sequences (IS) are important drivers of bacterial evolution. Here, we report a previously undescribed IS element (ISPst4) in Pseudomonas stutzeri, and its unusual interaction with plasmids introduced into this species. Transformation of the pUC19 derivative plasmid pUS23 into P. stutzeri yielded ampicillin-resistant transformants in P. stutzeri, but these grew very poorly. Plasmids recovered from the transformants frequently contained insertions of the IS elements ISPst4 and ISPst5. Hybridisation analysis showed that these two IS elements were common in P. stutzeri strains, but were not found in other pseudomonads. Insertions of ISPst4 in pUS23 were found predominantly between bla and oriV, and plasmids with this type of insertion were capable of robust replication in P. stutzeri, unlike pUS23. A promoter-containing region was localised to a 74 bp NcoI–SacI fragment within ISPst4, and we postulate that this promoter drives expression of the pUC oriV in P. stutzeri. This is the first report of IS transposition directly leading to an expansion of the effective host range of a plasmid, adding a new dimension to our understanding of the relationship between plasmids and IS elements.