Ensuring that one gene’s transcription does not inappropriately affect the expression ofits neighbors is a fundamental challenge to gene regulation in a genomic context. Inplants, which lack homologs of animal insulator proteins, the mechanisms that preventtranscriptional interference are not well understood. Here we show that BORDER proteins areenriched in intergenic regions and prevent interference between closely spaced genes on thesame strand by promoting the 3′pausing of RNA polymerase II at the upstream gene. In theabsence of BORDER proteins, 3′pausing associated with the upstream gene is reduced andshifts into the promoter region of the downstream gene. This is consistent with a model inwhich BORDER proteins inhibit transcriptional interference by preventing RNA polymerasefrom intruding into the promoters of downstream genes