This study investigates the role of microRNA (miRNA) in the toxicity of silver nanoparticles (SNPs) to human dermal fibroblasts (HDFs). First, 20 nm SNPs were synthesized, and the MTT assay of the influence of SNPs on HDF proliferation showed that cytotoxicity was not induced by 200 μM after 1, 4 and 8 h. Proteomics and miRNA sequencing technologies were then utilized to analyze protein and miRNA expression profiles, and 25 proteins and 246 miRNAs were found differentially expressed in HDFs treated with 200 μM SNPs. By integrating transcriptome, proteome and miRNA sequencing, 6 differentially expressed miRNAs were found regulated 3 target mRNA-protein pairs by inducing mRNA degradation and repressing protein translation, thus leading to differences in expression patterns. Bioinformatics analysis revealed that targeted mRNAs/proteins of SNP-induced differentially expressed miRNAs involved in 57 biological pathways. Four pathways were affected by differentially expressed miRNA, target mRNAs and target proteins simultaneously, namely 'Regulation of actin cytoskeleton,' 'Signaling of hepatocyte growth factor receptor,' 'Insulin signaling' and 'MAPK signaling pathway.' The results indicated that SNPs might induce HDF toxicity by affecting the cytoskeleton, ATP synthesis and apoptosis. The bioinformatics results were verified through cytoskeleton observation, ATP content analysis and cell apoptosis assay. SNP-induced differentially expressed miRNAs regulated the expression of target genes and proteins, leading to HDF toxicity through the destruction of cytoskeleton, reduction of intracellular ATP content and induction of apoptosis.