Objective The molecular mechanism of high-mobility group box 1(HMGB1) promoting the epithelial-mesenchymal transition (EMT) of gastric cancer(GC) has not been known well. This study aimed to explore the clinical effects of HMGB1 expression levels on the clinicopathological characteristics of patients with GC and to uncover the potential molecular mechanism by which promoting tumor progression. Methods The expression levels of HMGB1 in 125 pairs of patients with GC were detected by IHC and western blotting. Univariate and multivariate analyses were performed to evaluate the relationship between HMGB1 expression and clinical characteristics of patients with GC. Stable overexpression(Over-HMGB1) and knockdown(sh-HMGB1) GC cell lines(AGS and MKN-45) were used to determine the effects of HMGB1 on the activation of TLR4/NF-κB signaling. Differences were considered statistically significant at p<0.05 in two sides. Results HMGB1 is highly expressed in GC tissues and cell lines. High HMGB1 expression (HR=1.89, 95%CI:1.44-2.39, p=0.001) was an independent risk factor for OS in patients with GC. Downregulation of HMGB1 resulted in downregulation of TLR4 and NF-κB subunit (p-p65 and p-IκBα) expression, whereas the upregulated expression of HMGB1 led to increased expression of TLR4 and NF-κB subunits. Overexpression of HMGB1 promotes the upregulation of EMT-TF expression, which enhances the proliferation and migration abilities of GC cell lines. Conclusion HMGB1 is highly expressed in GC tissues and is associated with a poorer prognosis in patients with GC. HMGB1 activates the TLR4/NF-κB signaling pathway to promote EMT progression in GC cell lines. HMGB1 may be a critical molecule in prognosis prediction and a therapeutic target for patients with GC.