Abstract: As emerging environmental pollutants, antibiotic resistance genes (ARGs) and micro/nanoplastics (MPs/NPs) have been detected in various environmental media worldwide. Due to their small size and large surface area, MPs/NPs possess significant potential to act as carriers of ARGs. As such, the combined pollution of ARGs and MPs/NPs has attracted widespread attention due to their potential synergistic effects. Although MPs/NPs are considered key factors in the spread of ARGs in environmental media, knowledge of the effects and mechanisms of MPs/NPs on the horizontal transfer (particularly the transformation process) of ARGs in Escherichia coli (E. coli) is still largely limited. To bridge this knowledge gap, this study investigated the effects of different concentrations and sizes of polystyrene (PS) on the horizontal transfer of ARGs. We constructed a plasmid-mediated transformation system using the antibiotic-resistant plasmid pUC19 as donor and E. coli as recipient bacteria. Our results showed that after exposure to 5 mg/L 100 μm, 50 mg/L 100 μm, 5 mg/L 100 nm, and 50 mg/L 100 nm PS, the growth inhibition rates of E. coli reached 15.13%, 18.59%, 26.97%, and 35.84%, respectively (p < 0.01). In addition, the impact of PS MPs (≤ 5 mg/L) and PS NPs (≤ 50 mg/L) could significantly promote the transformation process of ARGs in a concentration-dependent manner. Under the same PS concentration, the ARGs transformation frequency decreased with increasing PS particle size. Specifically, 100 nm PS at 50 mg/L increased the transfer frequency by 79.34%. However, 1 mm PS particles at 50 mg/L resulted in a 21.80% decrease in the ARGs transformation frequency, suggesting that 1 mm PS particles at this concentration inhibit ARGs transfer. Based on the live/dead cell detection, we revealed that, with increasing PS concentration, cell membrane permeability in E. coli significantly increased by 56.66% to 69.47% compared to the control group. Under the condition of the same PS concentration, the cell membrane permeability rises as the PS size diminishes, implying a negative correlation between them. Similarly, the cell membrane permeability in NPs exposure was increased by 41.99% to 46.62%. These results demonstrated that high concentrations of NPs might enhance the cell membrane permeability in E. coli, thus facilitating the horizontal transfer of ARGs. Our findings elucidated the influence mechanism of MPs/NPs on the transformation of ARGs, providing a theoretical basis and technical guidance for the efficient assessments and control of combined ARGs and MPs/NPs pollution.