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The direct uptake of extracellular DNA (eDNA) via transformation facilitates the dissemination of antibiotic resistance genes (ARGs) in the environment. CeO₂ nanoparticles (NPs) have potential in the regulation of conjugation-dominated ARGs propagation, whereas their effects on ARGs transformation remain largely unknown. Here, CeO₂ NPs at concentrations lower than 50 mg L⁻¹ have been applied to regulate the transformation of plasmid-borne ARGs to competent <i>Escherichia coli</i> (<i>E. coli</i>) cells. Three types of exposure systems were established to optimize the regulation efficiency. Pre-incubation of competent <i>E. coli</i> cells with CeO₂ NPs at 0.5 mg L⁻¹ inhibited the transformation (35.4%) by reducing the ROS content (0.9-fold) and cell membrane permeability (0.9-fold), thereby down-regulating the expression of genes related to DNA uptake and processing (<i>bhsA</i>, <i>ybaV</i>, and <i>nfsB</i>, 0.7–0.8 folds). Importantly, CeO₂ NPs exhibited an excellent binding capacity with the plasmids, decreasing the amounts of plasmids available for cellular uptake and down-regulating the gene expression of DNA uptake (<i>bhsA</i>, <i>ybaV</i>, and <i>recJ</i>, 0.6–0.7 folds). Altogether, pre-exposure of plasmids with CeO₂ NPs (10 and 25 mg L⁻¹) suppressed the transformation with an efficiency of 44.5–51.6%. This study provides a nano-strategy for controlling the transformation of ARGs, improving our understanding on the mechanisms of nanomaterial-mediated ARGs propagation.