Protozoa, as primary predators of soil bacteria, represent an overlooked natural driver in the dissemination of antibiotic resistance genes (ARGs). However, the effects of protozoan predation on ARGs dissemination at the community level, along with the underlying mechanisms, remain unclear. Here we used fluorescence-activated cell sorting, qPCR, combined with metagenomics and reverse transcription quantitative PCR, to unveil how protozoa (Colpoda steinii and Acanthamoeba castellanii) influence the plasmid-mediated transfer of ARGs to soil microbial communities. Protozoan predation reduced the absolute abundance of plasmids but promoted the expression of conjugation-associated genes, leading to a 5-fold and 4.5-fold increase in conjugation frequency in the presence of C. steinii and A. castellanii, respectively. Excessive oxidative stress, increased membrane permeability, and the provoked SOS response closely associated with the increased conjugative transfer. Protozoan predation also altered the plasmid host range and selected for specific transconjugant taxa along with ARGs and virulence factors carried by transconjugant communities. This study underscores the role of protozoa in the plasmid-mediated conjugative transfer of ARGs, providing new insights into microbial mechanisms that drive the dissemination of environmental antibiotic resistance.