Abstract Purpose. The prognosis of advanced gastric cancer patients with peritoneal dissemination remains poor, and a better understanding of this mechanism is critical for development of new treatments that can improve patient survival. Long non-coding RNAs (lncRNAs) are most commonly defined as transcripts of greater than 200 nucleotides in length. Recent evidence suggests that lncRNAs are differentially expressed in cancer cells and have a major role in the development of cancer progression, including metastasis. This study aimed to investigate the clinical and biological significance of metastasis-associated lncRNA in gastric cancer. Experimental Design. We analyzed MALAT1 and HOTAIR expression levels by real-time reverse transcription PCR in 300 gastric tissues (150 gastric cancer tissues and 150 adjacent normal mucosa), and in seven gastric cancer cell lines. Knockdown of the HOTAIR by siRNA transfection was performed to evaluate HOTAIR function in gastric cancer cell lines and animal models. Results. Expression of MALAT1 and HOTAIR were significantly higher in cancerous tissues than in adjacent normal mucosa, and high expression of both lncRNAs significantly correlated with peritoneal metastasis in gastric cancer. In addition, elevated HOTAIR expression was an independent prognostic factor, and was an independent risk factor for peritoneal dissemination. SiRNA knockdown of HOTAIR in MKN45 and KATO-III cell lines resulted in reduced cell proliferation, colony formation, migration and invasion, but simultaneous increase in anoikis rates, compared to the controls. In an in vivo assay, HOTAIR siRNA-transfected MKN45 cells injected into nude mice inhibited the growth of xenograft tumors and peritoneal metastasis compared with scrambled siRNA-transfected cells. Conclusions. Our data highlight that HOTAIR expression may serve as a potentially important disease biomarker for the identification of high-risk gastric cancer patients. Moreover, our findings provide extensive mechanistic evidence for the functional role of HOTAIR over-expression and the ensuing malignant phenotype in gastric cancer cells in both cultured cells and two different animal models (xenograft and peritoneal metastasis). We propose that HOTAIR lncRNA could be used as a clinically useful diagnostic and prognostic biomarker for gastric cancer patients, and might be a novel therapeutic target for developing gastric cancer treatments. Citation Format: Yoshinaga Okugawa, Yuji Toiyama, Keun Hur, Shusuke Toden, Susumu Saigusa, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Masato Kusunoki, C. R. Boland, Ajay Goel. The expression of metastasis-associated long non-coding RNA, HOTAIR, is involved in cancer development and peritoneal metastasis in gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3553. doi:10.1158/1538-7445.AM2014-3553