Silver nanoparticles can be used to effectively disinfect Enterococcus faecalis (E. faecalis) in dental root canal therapy, of which the mechanism remains unclear. In the current study, we explored the effect of silver nanoparticles on bacterial growth and changes in the transcriptome of E. faecalis using RNA-sequencing technology. Results showed that silver nanoparticles significantly inhibited growth of E. faecalis by prolonging the lag phase in a dose dependent manner. A total of 100 differentially expressed genes (DEGs) including 3 upregulated and 97 downregulated DEGs were obtained in E. faecalis after treatment with silver nanoparticles. We performed Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses on these DEGs. Functional annotation of DEGs showed that transport and metabolism, such as carbohydrate metabolic process, peptide transport, and amide transport were most significantly enriched. Dysregulated genes were significantly enriched in pathways related to carbohydrate and energy metabolism, and the TCA cycle may be a major mechanism for E. faecalis with silver nanoparticle treatment. These data indicated changes in the transcriptome of E. faecalis after silver nanoparticle treatment, thus providing a mechanism for supporting application of silver nanoparticles for disinfection of E. faecalis during and after root canal therapy.