BACKGROUND & AIMS: Thousands of long intergenic non-protein coding RNAs (lincRNAs) have been identified in mammals via genome-wide sequencing studies. Many are functional, but are expressed aberrantly by cancer cells. We investigated whether levels of lincRNAs are altered during the development of esophageal squamous cell carcinoma (ESCC). METHODS: We used quantitative real-time polymerase chain reaction to measure levels of 26 highly conserved lincRNAs in ESCC and surrounding nontumor tissues. A total of 182 ESCC and paired adjacent nontumor tissue samples were collected from patients undergoing tylectomy at The First Affiliate Hospital of Soochow University from 2001 through 2009; another 178 ESCC tissue pairs were collected from Guangzhou Medical University from 2002 through 2009. LincRNAs were expressed from lentiviral vectors or knocked down with small hairpin RNAs in Eca-109 and TE-1 cells. RESULTS: Levels of a lincRNA encoded by a gene located next to POU3F3 (linc-POU3F3) were significantly higher in ESCC than neighboring nontumor tissues. In RNA immunoprecipitation assays, linc-POU3F3 was associated with the EZH2 messenger RNA (mRNA). Overexpression of linc-POU3F3 in cell lines increased their proliferation and ability to form colonies, and reduced the expression of POU3F3 mRNA, whereas knockdown of linc-POU3F3 increased the levels of POU3F3 mRNA. CpG islands in POU3F3 were densely hypermethylated in cell lines that overexpressed linc-POU3F3; methylation at these sites was reduced by knockdown of linc-POU3F3. Pharmacologic inhibition of EZH2 increased the levels of POU3F3 mRNA and significantly reduced binding of DNA methyltransferase (DNMT)1, DNMT3A, and DNMT3B to POU3F3. ESCC cells with knockdown of linc-POU3F3 formed xenograft tumors more slowly in mice than control ESCC cells. CONCLUSIONS: Levels of linc-POU3F3 are increased in ESCC samples from patients compared with nontumor tissues. This noncoding RNA contributes to the development of ESCC by interacting with EZH2 to promote methylation of POU3F3, which encodes a transcription factor.