Aeromonas veronii is a Gram-negative (G-) rod shaped facultative anaerobic bacteria of the family Aeromonadaceae. This study highlights the characterization of conjugation-related conjugative type IV secretion system (TFSS) from A. veronii MTCC3249, isolated from mosquito mid-gut. A. veronii harbours 3 plasmids, of which, pAc3249A was partially sequenced and sequence analysis revealed it carrying a typical TFSS. Thus far, this is the first functional, molecular and biochemical characterization of key components of TFSS encoded by a conjugative plasmid from Aeromonas. This study illustrated that the TFSS encoded on pAc3249A is functional and specific for its own oriT. The specificity could be correlated with the specific cleavage of the TraL. The TFSS of pAc3249A could mobilize the plasmid to G- bacteria but not to G+ bacteria. We also functionally characterized the key components such as three ATPases (TraE, TraJ and TraK), relaxase and its N-terminal domain (TraL and TraLN264 respectively). These studies provide basic information needed for further characterization of TFSS and its involvement in horizontal gene transfer and virulence of the bacteria.