The rapid dissemination of colistin resistance via mcr-carrying plasmids (pMCRs) poses a significant public health challenge. This study examined the genomic diversity and conjugation mechanisms of pMCRs, with a particular focus on the role of type IV secretion systems (T4SS) in IncI2 plasmids. The 868 complete plasmid sequences revealed various replicon types of pMCRs, with IncI2 as the primary epidemic type, and the co-transfer risk of multidrug resistance genes associated with IncHI2. T4SS was identified in 89.9% of pMCRs, with the T4SS sequence exclusively carried by IncI2 being conserved and typical of the VirB/D4 type, consisting of 12 subunits. Conjugation assays confirmed the essential role of the pilus subunit VirB2 and the significant impact of VirB5