Technical Abstract: Enhancer-promoter interactions potentially compromise the precise engineering of gene function and agronomically important traits in crops, which demands the adoption of strong, effective enhancer-blocking insulators to block such communication in a transgene construct. In this study, we evaluated and compared the insulation efficiency of three enhancer-blocking insulators using an enhancer activation assay in which a flower-specific AGIP promoter that drives a GUS reporter gene is activated in vegetative tissues by an adjacent CaMV35S enhancer. Of the insulators analyzed, only two of them, the EXOB fragment from the lambda genome and the TBS from petunia, are able to effectively block enhancer activation. Conversely, the 16 bp NI29 fragment from Arabidopsis inserted either as a single or a tandem repeat in the same assay vector failed as enhancer-blocking insulators as revealed by the detection of strong GUS expression in the leaves. PCR amplification and sequencing analyses showed that there were no deletion or mutation events detected within the NI29 sequence, indicating that the failure of NI29 in blocking 35S enhancer activation is not due to gene rearrangement or sequence mutation. Further analyses showed that the presence of EXOB or TBS in a transgenic unit did not influence either the stability of the transgene, or AGIP and CaMV35S promoter activity. This ruled out the possibility that the enhancer-blocking function of EXOB and TBS fragments resulted from the destabilization of the transgene or complete silencing of the AGIP or CaMV35S promoters’ activity. Quantitative analysis of GUS reporter expression at enzymatic and transcription levels exemplified that EXOB is at least four times more efficient than the TBS. The potential application of these insulators for remedying enhancer-promoter interactions in transgenes and possible mechanisms behind TBS and EXOB insulation function will be discussed.