Trichlorfon is a selective organophosphate insecticide that is widely applied in aquaculture and agriculture for control of various parasites. However, repeated and excess applications of trichlorfon often lead to water pollution and threaten non-targeted species. Our previous studies showed that trichlorfon could cause oxidative stress, lipid peroxidation and hepatic lesions in the liver of Rana chensinensis, but the related molecular mechanisms remain unclear. To explore the interference of trichlorfon in gene transcription, the differentially expressed genes in the liver of R. chensinensis exposed to trichlorfon were characterized using the RNA-seq platform. A search of all unigenes against non-redundant protein sequence (Nr), non-redundant nucleotide (Nt), Swiss-Prot, Kyoto Encyclopaedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG) and Gene Ontology (GO) databases resulted in 22,888, 21,719, 20,934, 16,923, 7375 and 15,631 annotations, respectively, and provided a total of 27,781 annotated unigenes. Among the annotated unigenes, 16,923 were mapped to 257 signalling pathways. A set of 3329 differentially expressed unigenes was identified by comparison of the two groups in liver. Notably, relative expression of metabolism-related genes, including both up and down-regulated genes, were also validated by qPCR. The present study depicts the high degree of transcriptional complexity in R. chensinensis under trichlorfon stress and provides new insights into the molecular mechanisms of organophosphate insecticide toxicology. Some of these metabolism-responsive genes could be useful for understanding the toxicological mechanism of trichlorfon on non-target aquatic organisms and will contribute to the conservation of aquatic life.