Convolvulus arvensis is a troublesome weed that is naturally tolerant to glyphosate. This weed tolerates glyphosate at a rate 5.1 times higher than that of glyphosate-susceptible Calystegia hederacea. Glyphosate-treated C. arvensis plants accumulated less shikimic acid than C. hederacea plants. The overexpression of EPSPS genes from the two species in transgenic Arabidopsis thaliana resulted in similar glyphosate tolerance levels. qPCR of genomic DNA revealed that the EPSPS copy number in C. arvensis was approximately 2 times higher than that in C. hederacea. Moreover, glyphosate treatment caused a marked increase in EPSPS mRNA in C. arvensis compared to C. hederacea. GUS activity analysis showed that the promoter of CaEPSPS (CaEPSPS-P) highly improved GUS expression after glyphosate treatment, while no obvious differential GUS expression was observed in ChEPSPS-P transgenic A. thaliana in the presence or absence of glyphosate. Based on the obtained results, two coexisting mechanisms may explain the natural glyphosate tolerance in C. arvensis: (i) high EPSPS copy number and (ii) specific promoter-mediated overexpression of EPSPS after glyphosate treatment.