I-complex plasmids (IncB/O, IncK1, IncK2, IncI1, IncI2, IncIÉ£, IncZ) are associated with pathogenic Enterobacteriaceae and contribute to antibiotic resistance. The replication and transfer regions of I-complex plasmids are similarly organized but demonstrate low sequence conservation between subgroups. To date, only the IncI1 plasmid R64 has been comprehensively characterized. This study investigated pMS7163B, an I-complex plasmid carrying an IncZ replicon on an IncB/O backbone that is distantly related to R64. Plasmid pMS7163B was subjected to in vitro transposon mutagenesis and subsequent transposon-directed insertion site sequencing (TraDIS), which identified genes required for replication/maintenance (following transformation into E. coli TOP10) and surface conjugation (following conjugation to E. coli J53). The replicon region and a partitioning system parAB were required for pMS7163B replication/maintenance. Thirty-five genes were required for surface conjugation, revealing a role for the tra genes (encoding a type IV secretion system necessary for DNA transfer) but not the pil genes (encoding a long-thin pilus necessary for cell-cell contact in liquid mating). Two genes (ardB and 910) demonstrated donor-specific roles possibly linked to methylation, reducing pMS7163B conjugation frequency from a TOP10 but not an MG1655 donor background when mutated. Additional detailed analysis of the TraDIS data identified several highly conserved genes (impABC, 810, parB_2, 930/940) that contained an upstream strand-specific transposon insertion bias. We demonstrated that overexpression of these genes resulted in either a severe growth defect of the host cell or rapid out-competition within a mixed population. Thus, rather than playing a specific role in conjugation, these genes, which are broadly conserved I-complex plasmids, must be tightly regulated to avoid deleterious impacts on stability and maintenance. Deciphering the function of these genes and their role in different hosts will enhance our understanding of the epidemiology of I-complex plasmids and their impact on increasing antibiotic resistance.