Pyroligneous acids (PAs) amendments could reduce soil antibiotic resistance genes (ARGs) pollution, but their impacts on horizontal transformation of extracellular ARGs (eARGs) remain unclear. Here, a wood residues derived PA was selected to investigate its effect on ARG dissemination via transformation using a soil microcosm experiment and an in vitro transformation system. The PA application effectively decreased the abundances of representative ARGs and mobile genetic elements, demonstrating that the weakened horizontal gene transfer alleviated ARG pollution in the soil. PA showed an amount-dependent inhibition on the transformation as well as the three distilled fractions and chemical components, proving that their important roles in inhibiting eARG transformation. The relatively low-amount (1 mu L mL-1) of PA suppressed the transformation mainly by destroying the plasmid pBR322 structure, while the high-amount (10-100 mu L mL-1) of PA inhibited the transformation due to the inactivation of recipient Escherichia coli DH5 alpha by inducing oxidative stress and destroying cell membrane, and damages of plasmid by reducing eARGs abundance and broking the base deoxyribose, and phosphate skeletons. These findings expand the understanding of PA amendments mitigating ARG pollution in agricultural soils via inhibiting horizontal gene transformation, and also provide a practical strategy to remediate soil ARG pollution.