Plasmids 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 in Pasteurellaceae, 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 in E. coli cells, elevating the stability, and resistance profile. The second mechanism consists of two insertion sequences (ISs), IS1 and IS10, which decrease the fitness cost of the plasmid by disrupting an uncharacterized gene on pB1000 that is harmful to E. 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 in oriV prevent 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 IS1 leads to expansion, IS10 cannot 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. IMPORTANCE ColE1-like plasmids are small, mobilizable plasmids that can be found across at least four orders of Gammaproteobacteria and are strongly associated with antimicrobial resistance genes. Plasmid pB1000 carries the gene blaROB-1, conferring high-level resistance to penicillins and cefaclor. pB1000 has been described in various species of the family Pasteurellaceae, for example, in Haemophilus influenzae, which can cause diseases such as otitis media, meningitis, and pneumonia. To understand the resistance spread through horizontal transfer, it is essential to study the mechanisms of plasmid adaptation to novel hosts. In this work we identify that a gene from pB1000, which encodes a peptide that is toxic for E. coli, and the low plasmid copy number (PCN) of pB1000 in E. coli cells are essential targets in the described plasmid-host adaptation and therefore limit the spread of pB1000-encoded blaROB-1. Furthermore, we show how the interplay of two adaptation mechanisms leads to successful plasmid maintenance in a different bacterial family.