Long non-coding RNAs (lncRNAs) have been shown to epigenetically regulate certain genes in human cells. Here we report evidence for the involvement of an antisense lncRNA in the transcriptional regulation of the pluripotency-associated factor Oct4. When an lncRNA antisense to Oct4-pseudogene 5 was suppressed, transcription of Oct4 and Oct4 pseudogenes 4 and 5 was observed to increase. This increase correlated with a loss of silent state epigenetic marks and the histone methyltransferase Ezh2 at the Oct4 promoter. We observed this lncRNA to interact with nucleolin and PURA, a 35 kD single-stranded DNA and RNA binding protein, and found that these proteins may act to negatively regulate this antisense transcript.