ABSTRACT Dermatophagoides farinae is a major source of allergens that are the cause of allergic diseases in humans. Methyl isoeugenol (MIE) is a naturally occurring compound in plants and its strong acaricidal toxicity has been established; however, the mechanism behind its acaricidal properties remains unclear. In this study, to investigate the action mechanism of MIE on D. farinae the isobaric tags for relative and absolute quantification (iTRAQ) proteomics method is used. Protein samples were extracted from D. farinae before and after 24 h of MIE treatment for iTRAQ technique analysis. Bioinformatics analysis revealed that most differentially expressed proteins were located in the cytoplasm, impacting cytoplasmic function. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed significant enrichment of the differential proteins in the cellular autophagy signalling pathway. The autophagy-related proteins Autophagy-Related Protein 3 (ATG3) and Cathepsin L (CTSL) were up-regulated, disrupting the autophagic process. We found that the main action mechanism of MIE against D. farinae might be cellular autophagy. The transcriptional validation of six differential proteins demonstrated consistent expression trends with the proteomics data. This study reveals the acaricidal mechanism of MIE for the first time and lays the foundation for further research on D. farinae control.