Background <p><i>Chrysomya megacephala</i> is a forensically important dipteran that significantly facilitates the estimation of Post Mortem Interval (PMI) in forensic entomology. In this study, we investigated the sublethal effects of monocrotophos, a widely used insecticide, on third-instar larvae of <i>Chrysomya megacephala</i>.</p> Methods and results <p>The EC₅₀ value for pupation inhibition was determined to be 0.23 μg/ml, and the 1/3rd of EC₅₀ concentration (0.077 μg/ml) was selected for studying the pesticide-induced effects on pupation time and for the de novo transcriptome analysis. The pupation time was determined to understand the impact of monocrotophos on developmental rate and PMI calculation. As there is no previously published genome of this species or any closely related species, the de novo transcriptomic profiling of the third-instar larvae was performed to determine the effects on pupation and other biological aspects. The statistical analysis revealed a significantly accelerated pupation rate in Treatment (I), compared to Control (C), and the transcriptomic profiling showed the differential expression of numerous genes supporting this observation, indicating that monocrotophos may potentially disrupt the accurate determination of PMI.</p> Conclusions <p>Molecular expression profiling showed 17,865 Differentially Expressed Genes, among which 8,731 were downregulated, and 9,134 were upregulated. Functional annotation (KOG) and pathway enrichment analysis (GO and KEGG) revealed that the effect of monocrotophos alters the expression of a significant number of genes involved in various biological processes, including amino acid, carbohydrate, and lipid transportation and metabolism; proteolysis; monooxygenase activity; regulatory RNA binding; regulation of gene expression; signal transduction; xenobiotics biodegradation and metabolism; and glycan biosynthesis and metabolism.</p>