High mobility group box 1 (HMGB1) as a novel inflammatory molecule has been shown to be involved in a variety of cell physiological and pathological behaviors including immune response, inflammation and cancer. Evidence suggests that HMGB1 plays a critical role in the development and progression of multiple malignancies. However, the underlying molecular mechanisms for the HMGB1-mediated growth and invasion of gastric cancer have not yet been elucidated. The present study investigated the expression of HMGB1 in gastric adenocarcinoma (GAC) and the mechanisms by which it contributes to tumor growth and invasion. The correlation between HMGB1 expression and clinicopathological characteristics of GAC patients was assessed by immunohistochemical assay through tissue microarray procedures. The RNA and protein expressions of HMGB1 and downstream factors were detected by quantitative PCR and western blot assays; cell proliferation and invasion were determined by MTT, wound-healing and 3D-Matregel assays, subcutaneous SGC-7901 tumor models were established to verify tumor growth in vivo. We demonstrated that, the expression of HMGB1 was significantly increased in the nucleus of GAC tissues compared with that in adjacent non-cancer tissues (88.6 vs.70.5%, P<0.001), and correlated with the metastatic lymph node of GAC (P=0.018). Furthermore, knockdown of HMGB1 by shRNA inhibited cell proliferative activities and invasive potential, and downregulated the expression of NF-κB p65, PCNA and MMP-9 in GAC cells (SGC-7901 and AGS). The tumor volumes in SGC7901 subcutaneous nude mouse models treated with Lv-shHMGB1 was significantly smaller than those of the nonsense sequence group. Taken together, these findings suggest that increased expression of HMGB1 is associated with tumor metastasis of GAC, and knockdown of HMGB1 suppresses growth and invasion of GAC cells through the NF-κB pathway in vitro and in vivo, suggesting that HMGB1 may serve as a potential therapeutic target for GAC.