As emerging pollutants, antibiotic resistance genes (ARGs) and microplastics threaten the environment and human health. Gut microbiota is a hotspot for ARG emergence and spread. However, effects of microplastic exposure on the emergence and spread of gut microbial ARGs are unclear. Therefore, metagenomics was used to characterize polystyrene microplastics (PS)-induced ARG alterations in rat gut microbiota and their health risks, and to identify key ARG hosts and pathways as intervention targets. We found that PS exposure not only induced selective accumulation of glycopeptide and aminoglycoside ARGs, but also promoted mobility risks of glycopeptide and macrolide-lincosamide-streptogramin ARGs in gut microbiota. Metagenomic reassembly identified microbes belonging to Firmicutes (particularly order Clostridiales, such as speices Lachnospiraceae bacterium 3-1 and MD335) as major ARG hosts. Meanwhile, genera Enterococcus, Clostridioides and Streptococcus were main ARG hosts among human pathogens. Furthermore, glycopeptide and aminoglycoside ARGs were highly correlated with VanS/VanR signaling and its regulatory pathways of vancomycin resistance and peptidoglycan metabolism, amino sugar and nucleotide sugar metabolism, and CpxR signaling and its regulatory remodeling of cell envelope peptidoglycans and proteins in gut microbiota upon PS exposure. This study provides novel insights and intervention targets involved in PS-induced changes in gut microbial ARGs and their health risks.