Ethyl formate has been used for the control of insect pests by fumigation. However, there were not many reports toshow its target site of fumigant toxicity on insect pests since its first use in the agricultural industry. In the present study,we showed the presumable target sites of ethyl formate fumigation in insect pests using Myzus persicae nymphs. Afterethyl formate fumigation, the nymphs of this species were collected and the changes at the biochemical and molecularlevel were determined. The activity of cytochrome c oxidase (COX) was approximately two-fold higher after ethyl formatefumigation. In addition, the expression levels of acetylcholinesterase (AChE) decreased gradually with increasing ethylformate concentration. These two findings suggested that COX and AChE might be the major target sites of ethyl formatefumigation. In addition to these results, the analysis of lipid content using MALDI-TOF MS/MS identified 9 phospholipidsdifferently generated 2-fold higher in the ethyl formate-treated nymphs than that in the control nymphs, thereby leadingto changes in cell membrane composition in M. persicae nymphs. Therefore, the ethyl formate fumigation caused lethaleffects on M. persicae nymphs by changing COX activity, AChE gene expression, and phospholipid production.