Differential expression of LINC00339 is involved in the malignancy of multiple human cancer types. Nonetheless, the expression profile, functions, and potential mechanisms of action of LINC00339 in gastric cancer are yet to be fully elucidated. This study aimed at measuring LINC00339 expression in gastric cancer and examining the prognostic significance of LINC00339 in patients with gastric cancer. The detailed functions of LINC00339 with regard to the aggressive characteristics of gastric cancer cells and the underlying molecular mechanisms were investigated. Here, we found that LINC00339 expression was aberrantly high in gastric cancer and significantly associated with lymph node metastasis, invasive depth, and TNM stage. Patients with gastric cancer in a LINC00339 high-expression group showed shorter overall survival than patients in a LINC00339 low-expression group. A knockdown of LINC00339 suppressed gastric cancer cell proliferation, migration, and invasion and induced apoptosis in vitro and slowed tumor growth in vivo. In terms of the mechanism, LINC00339 was found to act as a molecular sponge on microRNA-539 (miR-539). SRY-box 9 (SOX9) was confirmed as a direct target gene of miR-539 in gastric cancer cells. An miR-539 knockdown attenuated the effects of the LINC00339 knockdown on the malignant characteristics of gastric cancer cells. Overall, LINC00339 plays a critical role in the malignancy of gastric cancer by regulating SOX9 via sponging of miR-539. Our findings highlight the importance of the LINC00339-miR-539-SOX9 pathway in gastric cancer pathogenesis and may point to novel targets for the diagnosis, prognosis, and/or treatment of gastric cancer.