ABSTRACTPlasmids facilitate the vertical and horizontal spread of antimicrobial resistance genes between bacteria. The host range and adaptation of plasmids to new hosts determine their impact on the spread of resistance. In this work, we explore the mechanisms driving plasmid adaptation to novel hosts in experimental evolution. Using the small multicopy plasmid pB1000, usually found inPasteurellaceae, we studied its adaptation to a host from a different bacterial family,Escherichia coli. We observed two different mechanisms of adaptation. One mechanism is single nucleotide polymorphisms (SNPs) in the origin of replication (oriV) of the plasmid, which increase the copy number inE. colicells, elevating the stability, and resistance profile. The second mechanism consists of two insertion sequences (ISs), IS1and IS10, which decrease the fitness cost of the plasmid by disrupting an uncharacterized gene on pB1000 that is harmful toE. coli. Both mechanisms increase the stability of pB1000 independently, but only their combination allows long-term maintenance. Crucially, we show that the mechanisms have a different impact on the host range of the plasmid. SNPs inoriVprevent the replication in the original host, resulting in a shift of the host range. In contrast, the introduction of ISs either shifts or expands the host range, depending on the IS. While IS1leads to expansion, IS10cannot be reintroduced into the original host. This study gives new insights into the relevance of ISs in plasmid-host adaptation to understand the success in spreading resistance.