Abstract Background: Long noncoding RNAs (lncRNAs) have been illustrated to function as important regulator in carcinogenesis and cancer progression. However, the roles of lncRNA NNT-AS1 in gastric cancer remain unclear. In present study, we investigate the expression level of NNT-AS1 in gastric cancer tissue and the role on gastric cancer cells tumorigenesis. Methods: NNT-AS1 expression level was measured using RT-PCR. The prognosis and survival rate of gastric cancer patients with high/low expression of NNT-AS1 was tracked and analyzed. Cellular experiments were performed to test the role of NNT-AS1 on gastric cancer phenotype, including proliferation analysis, transwell assay, EMT-related marker blots and xenograft mice assay. Bioinformatics online program and luciferase assay were performed to predict and verify the potential miRNAs targeting NNT-AS1. Results: NNT-AS1 expression level was significantly upregulated in 55 cases of gastric cancer tissue samples compared with adjacent normal tissue. Besides, the aberrantly enhanced NNT-AS1 expression predicted poor prognosis and lower survival rate. In vitro, NNT-AS1 knockdown suppressed the gastric cancer cells proliferation, invasion and EMT-related marker (N-cadherin and vimentin) expression. In vivo, lentivirus mediated NNT-AS1 silencing decreased the tumor growth. MiR-503 was verified to be the target miRNA of NNT-AS1 using bioinformatics online program and luciferase assay. Furthermore, miR-424 could reverse the role of NNT-AS1 on gastric cancer cells. Conclusion: In conclusion, our study indicates that NNT-AS1 sponges miR-424 to facilitate gastric cancer cells invasion and metastasis, revealing the oncogenic role of NNT-AS1 on gastric cancer cells. Citation Format: Beibei Chen, Qingfang Zhao, Lulu Guan, Huifang Lv, Liangyu Bie, Jinxi Huang, Jitian Li, Jianying Zhang, Xiaobing Chen. Long noncoding RNA NNT-AS1 sponges miR-424 to promote the progression and metastasis of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2475.