Objective To identify differentially expressed genes (DEGs) in Dermatophagoides farinae post-treatment with horseradish using RNA sequencing, and to unravel the underlying mechanisms. Methods Ninety fumigation bottles were randomly divided into the experimental and control groups. In each bottle, 80 D. farinaes were placed back down on the adhesive plates of the fumigation bottles. D. farinaes were exposed to 25 μl of 0.25 ml/L horseradish dilutions in the experimental group, and paraffin in the control group for 24-hour fumigation. Then 2 000 survival D. farinae were collected from each group. All experiments were repeated 6 times, with triplicates for sequencing and others for real-time fluorescent quantitative PCR (qPCR) verification. RNA was extracted from D. farinae and reversely transcribed into cDNA, and a library was constructed for RNA sequencing. Following quality control and assembly, the sequencing data were aligned with non-redundant protein sequence (NR), gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) databases, and the GO and KEGG functional annotations and enrichment analysis of DEGs were performed with the edge R software. In addition, DEGs identified in D. farinaes post-treatment with horseradish were sampled for qPCR assay, the qPCR data were subjected to analysis of variance with the R software. Results RNA sequencing yielded 56 625 transcripts with a total length of 72 418 610 nt, and a total of 39 510 UniGene clusters were yielded, with the highest number of clusters aligned to the NR database (25 450 clusters), followed by the evolutionary genealogy of genes: non-supervised orthologous groups (eggNOG） database (17 643 clusters), Swiss-prot database (13 338 clusters), GO database (13 234 clusters), and KEGG database (9 445 clusters), respectively. A total of 2 719 DEGs were identified in D. farinae post-treatment with horseradish, including 1 185 up-regulated genes and 1 534 down-regulated genes. GO annotations showed that the DEGs were mainly related to cellular processes, binding and catalytic activities, and GO enrichment analysis showed that the up-regulated genes were mainly enriched in ATP-binding cassette transporter G (ABCG) subfamily, cytochrome P450 (CYP450), heat shock proteins, and acetylcholine receptor and the down-regulated genes were mainly enriched in calmodulin and RNA polymerase. KEGG annotations showed that DEGs were mainly involved in the processes of transport and catabolism, signal transduction, and translation, and KEGG enrichment analysis revealed that the DEGs were significantly enriched in 30 pathways, which were mainly enriched in ribosome, microbial metabolism across diverse environments, and biosynthesis of secondary metabolites. qPCR results show that the relative transcription levels of UDP-glucuronosyltransferase (UGT) and multidrug resistance-associated protein 1 (MRP1) in the experimental group were 1.18 ± 0.22 and 6.22 ± 0.42, respectively, which were significantly upregulated compared to the control group (0.76 ± 0.30 and 2.52 ± 1.75) (F = 11.22, 16.83, both P < 0.05). And the relative transcription level of mitochondrial ribosome-associated GTPase (MTG) was 0.05 ± 0.04, which was significantly downregulated compared to the control group (1.23 ± 0.62) (F = 13.95, P < 0.05), which was consistent with RNA sequencing results. Conclusion There is differential expression of multiple genes in D. farinae post-treatment with horseradish, and UGT, CYP450, MRP1 and ABCG genes play important roles in the development of resistance to horseradish or detoxification metabolism of horseradish.