The high mobility group box-B1 (HMGB1)-receptor for advanced glycation end-products (RAGE) and the protein kinase B (Akt) pathways play a crucial role in tumorigenesis and development of many malignant tumors. Ethyl pyruvate (EP), a potent inhibitor of HMGB1 release, can exert antitumor effects on the growth of gastric cancer. Therefore, it is necessary to observe the effects of EP on gastric cancer growth in vitro and in vivo. Human gastric adenocarcinoma tissues of different grades (N=45) were collected. The expression of HMGB1 and RAGE was evaluated immunohistochemically in biopsy samples. After SGC-7901 gastric cancer cells were treated with EP, the expression of HMGB1, RAGE, Akt, phosphorylated Akt (p-Akt) and some transcription factors was identified and the effects of EP on cell proliferation, invasion, cell cycle distribution and apoptosis were assessed. A subcutaneous xenograft tumor model was established, validating the effects of EP on tumor growth in vivo. The expression of HMGB1 and RAGE was respectively observed in 73.3 and 68.9% of the gastric adenocarcinoma tissues. The frequency of positive expression increased with the ascending grade of the tumor malignancy. EP decreased the expression of HMGB1, RAGE, Akt, p-Akt, Ki-67 and matrix metallopeptidase-9 (MMP‑9), and increased the expression of p53. Moreover, EP could inhibit tumor cell proliferation and invasion, induce cell cycle arrest and apoptosis and slow the growth of xenograft tumors. In conclusion, HMGB1 and RAGE were strongly expressed in gastric adenocarcinoma, and EP administration inhibited gastric cancer growth via regulation of the HMGB1-RAGE and Akt pathways. EP may play a critical role in the treatment of cancer in conjunction with other therapeutic agents.