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Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and overexpression of the EPSPSgene is one of the molecular mechanisms conferring glyphosate resistance in weeds. A regulatory sequence of EPSPSgene was isolated previously, and an alteration in its 5´-untranslated region (UTR) pyrimidine (Py)-rich stretch element is involved in the regulation of EPSPSexpression in glyphosate-resistant (GR) Eleusine indica.However, the transcription factors involved in this regulatory sequence remain to be elucidated. In this study, we investigated the regulatory network of EPSPSoverexpression associated genes in a GR E. indicapopulation by RNA-seq. The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2752 unigenes and a decrease in the expression of 4025 unigenes in the GR E. indica,compared to the glyphosate-susceptible (GS) E. indica.Among them, 1373 unigenes were identified to be co-expressed with the EPSPSgene in GR E. indica.GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway, chlorophy II and peroxisome metabolism processes. Notably, the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate (S3P, a substrate of EPSPS), was also up-regulated. Eight transcription factors were identified as likely to be involved in the regulation of the EPSPSexpression, and three of them (ARF2, ARF8and BPC6) showed more binding sites because of a (CT)ninsertion of the 5´-UTR Py-rich stretch element in GR. However, the yeast one-hybrid assay illustrated that ARF8and BPC6could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS,but could not bind to the mutated form. Our data suggests that the transcriptional regulation of EPSPSexpression is complex and was significantly altered in GR E. indica.These discoveries provide new references for further study of the EPSPSoverexpression mechanism that endows glyphosate resistance.