Ebselen is a seleno-organic compound that has been demonstrated to have antioxidant and anti-inflammatory properties. A previous study determined that ebselen inhibits airway inflammation induced by inhalational lipopolysaccharide (LPS), however, the underlying molecular mechanism remains to be elucidated. The present study investigated the effect of ebselen on the glutathione peroxidase (GPX)-reactive oxygen species (ROS) pathway and interleukin-8 (IL-8) expression induced by Helicobacter pylori LPS in gastric cancer (GC) cells. Cells were treated with 200 ng/ml H. pylori-LPS in the presence or absence of ebselen for various durations and concentrations (mu mol/l). The expression of toll-like receptor 4 (TLR4), GPX2, GPX4, p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated-p38 MAPK, ROS production and IL-8 expression were detected with western blotting or ELISA. The present study revealed that TLR4 expression was upregulated; however, GPX2 and GPX4 expression was reduced following treatment with H. pylori LPS, which led to increased ROS production, subsequently altering the IL-8 expression level in GC cells. Additionally, it was determined that ebselen prevented the reduction in GPX2/4 levels induced by H. pylori LPS, however, TLR4 expression was not affected. Ebselen may also block the expression of IL-8 by inhibiting phosphorylation of p38 MAPK. These data suggest ebselen may inhibit ROS production triggered by H. pylori LPS treatment via GPX2/4 instead of TLR4 signaling and reduce phosphorylation of p38 MAPK, resulting in altered production of IL-8. Ebselen may, therefore, be a potential therapeutic agent to mediate H. pylori LPS-induced cell damage.