[Objectives]  To investigate stress gene expression, and quantitative changes in the expression of this gene, in Drosophila S2 cells treated with parathion, and provide a basis for further analysis of insect stress reactions and molecular toxicology. [Methods]  The cytotoxicity and cell survival rate of Drosophila S2 cells were tested using CC-8 reagents and a cell count plate. Application of RNA-Seq and qRT-PCR to detect differentially expressed genes and quantitative changes in gene expression. [Results]  Cells exposed to 35 μg/mL parathion had high survival rates. Analysis of changes in gene expression with RNA-Seq revealed that 481 genes were up-regulated and that 52 displayed significant changes. These included oxidative stress genes, detoxification genes and immune related genes. [Conclusion]  Identifying genes that are differentially expressed in response to cytotoxicity lays the foundation for analyzing the acting targets of parathion and the mechanism underlying resistance to this pesticide.