IncA/C plasmids are broad host-range and can spread readily between bacteria of different genera via conjugation. They are increasingly associated with dissemination of antibiotic resistance genes including those conferring resistance to last resort drugs such as carbapenems (blaNDM-1) and third-generation cephalosporins (blaCMY). Although conjugation is a major underlying mechanism of the IncA/C-mediated dissemination of antibiotic resistance, the genetics of IncA/C conjugation is still poorly understood, with only a few genes experimentally shown to be involved in conjugation. In this study, we utilised hyper-saturated transposon mutagenesis coupled with transposon directed insertion site sequencing (TraDIS) to determine the set of genes required for conjugation of the prototype IncA/C plasmid pMS6198A in E. coli. Our analysis identified all previously known and predicted conjugation genes. In addition, we also identified several new genes required for conjugation, each of which was independently confirmed by targeted mutagenesis and conjugation assays. Interestingly, we have confirmed the role of a previously uncharacterised regulator (referred to as acaB), which acts at the top of the hierarchical cascade of conjugation control. Our data showed that AcaB activates conjugation via upregulation of AcaDC, a master regulator that in turn directly activates the transcription of conjugation genes. Taken together, this work describes the most complete set of IncA/C conjugation genes to date and sheds light on a key regulatory step in the IncA/C conjugation control pathway. The genes identified in this work could be the targets of novel molecules to inhibit plasmid conjugation and thus reduce the spread of antibiotic resistance determinants.