This study aimed to elucidate the roles of long non-coding RNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) in gastric cancer. The expression of NEAT1 in gastric cancer tissues and cells was determined. NEAT1 was then overexpressed and suppressed in BGC-823 cells in vitro to further explore the effects of NEAT1 on cell proliferation, migration, and invasion. In addition, the regulatory relationship among NEAT1, miR-335-5p and Rho Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1) was investigated. LncRNA NEAT1 was upregulated in gastric cancer tissues and cells. Upregulated NEAT1 significantly promoted the proliferation, migration and invasion of BGC-823 cells, while suppression of NEAT1 exhibited contrary results. In addition, lncRNA NEAT1 inhibited the expression of miR-335-5p, and miR-335-5p targeted ROCK1 in BGC-823 cells. miR-335-5p overexpression significantly inhibited cell proliferation, migration and invasion, which was counteracted by ROCK1 overexpression concurrently. Our findings indicate that upregulation of NEAT1 may promote proliferation, migration and invasion of gastric cancer cells via targeting miR-335-5p/ROCK1 axis. NEAT1-miR-335-5p-ROCK1 axis may be a potential therapeutic strategy for gastric cancer therapy.