ABSTRACT: Glyphosate (GLY) is one of the most applied agrochemicals in the world, and its exposure has aroused the public health concern. Freshwater planarian is an ideal test organism for examining toxicity of pollutants and has become an emerging animal model for toxicology studies. However, potential toxicity mechanism of GLY in planarian has not been comprehensively studied. To explore the toxicity effects and molecular mechanism involved in GLY exposure of planarian, we analyzed the acute toxicity, histological change, DNA damage and transcriptional responses of freshwater planarian Dugesia japonica subjected to GLY. Results indicated that morphological malformation, histopathological change and genome DNA damage were observed after GLY exposure for different time. Transcriptomic analysis showed that numerous differentially expressed genes (DEGs) were obtained at 1, 3 and 5 d after exposure. GO and KEGG enrichment analysis of these DEGs was carried out and a dynamic and global view was obtained in planarian after GLY exposure at the transcriptomic level. Furthermore, qRT-PCR was performed on nine DEGs associated with detoxification, apoptosis, stress response, DNA repair, etc. The expression patterns were well consistent with the RNA-seq results at different timepoints, which confirmed the accuracy and reliability of the transcriptome data. Collectively, our results established that GLY could pose adverse effects on the morphology, histoarchitecture as well as genome DNA of D. japonica, and the planarians are capable of responding to the disadvantageous GLY stress by dysregulating the related genes and their participated in pathways concerning immune response, detoxification, energy metabolism, DNA damage repair, etc. To the best of our knowledge, this is the first report of transcriptomic analysis of freshwater planarian exposed to environmental pollutant, and it provided detailed sequencing data deriving from transcriptome profiling to deepen our understanding of the molecular toxicity mechanism of GLY on planarians.