CCCTC-Binding Factor (CTCF) is an architectural protein that acts as an insulator element and is enriched at transitions between euchromatin and heterochromatin1. It has been demonstrated previously that removal of CTCF from these locations can lead to aberrant spreading of H3K4me3 and activation of silenced genes2,3. CTCF is also localised at gene promoters, which led us to hypothesise that CTCF may act as an insulator of bidirectional transcription. To investigate the potential insulator function of CTCF at bidirectional gene promoters we performed RNAi knockdown of CTCF followed by gene expression profiling in prostate cancer cell line, LNCaP. Following CTCF knockdown we found that of the 155 genes that had CTCF bound at their promoters and were changed in gene expression, prior to knockdown 93% (145/155) lost this binding after CTCF depletion. To assess whether CTCF promoter depletion was accompanied by changes in levels of H3K4me3, we also performed H3K4me3 ChIP-seq following CTCF RNAi. We found that 41% (60/145) of gene promoters that lost CTCF also had altered levels of H3K4me3. Interestingly 25% of these (15/60) genes demonstrated ‘spreading’ of H3K4me3 across a bidirectional promoter and an increase in transcription of the neighbouring gene. An additional 16% (10/60) of genes showed spreading of H3K4me3 away from a unidirectional promoter into the neighbouring intergenic region and a decrease in expression of the underlying gene. This data provides preliminary evidence that CTCF may aid in the regulation of gene expression by 1. Restriction of H3K4me3 nucleosomes at unidirectional promoters and 2. Insulation of aberrant bidirectional transcription by impeding the spreading of H3K4me3 associated nucleosomes.