With the rapid development of nanotechnology, nanomaterials have been prepared with diverse sizes and surface properties. However, the influences of these properties on the horizontal transfer of antibiotic resistance genes (ARGs) are still unclear. Here, two groups of Ag particles were prepared, one group with the same negative charge and different sizes (9, 72 and 1885 nm), the other group with similar sizes (72 and 67 nm) and different charges (−12.7 and +22.6 mV) and their effects on ARGs transformation were investigated. The results revealed that 9 nm Ag at 1000 μg/L induced a 14.56-fold increase in transformation, which was 2.05 and 3.91 times higher than that of 72 nm and 1885 nm Ag particles, respectively. When the size was similar, the positively charged Ag stimulated an 11.59-fold increase in transformation, which is 1.63-fold higher than that of the negatively charged Ag. Investigation of the underlying mechanisms suggested that Ag particles with a smaller size and/or positive charge induced more intercellular reactive oxygen generation and membrane damage than Ag particles with large size and/or negative charge. This study exposed the potential ecological risks of Ag particles with small size and positive charge in accelerating the dissemination of ARGs and highlighted concerns about the management of nanomaterials with varying structural properties.