As an inhibitor of high mobility group protein B1 (HMGB1), ethyl pyruvate (EP) has been associated with various inflammatory diseases. Recent studies have investigated the relationship between EP and cancer. The present study aimed to determine the antitumor efficacy of EP in non‑small cell lung cancer (NSCLC) cells and elucidate the underlying mechanism. A549, H520 and PC‑9 cells were treated with EP in suitable concentrations. RT‑qPCR and western blot analysis were performed to evaluate HMGB1 and RAGE expression levels. MTT and colony formation assays assessed the effect of EP on cell growth. A Transwell assay was used to evaluate invasion and migration potential and flow cytometry was performed to analyze cell apoptosis. Bcl‑2 family proteins were identified by western blot analysis. The results demonstrated that an increased EP concentration effectively reduced HMGB1 and RAGE expression, thus inhibiting the HMGB1/RAGE axis. EP decreased level of PCNA and MMP‑9 and increased P53 levels. Bcl‑2 and Mcl‑1 were also decreased, whereas Bax expression was increased. Furthermore, a high concentration of EP (30 mmol/l) significantly inhibited NF‑κB and STAT3 activation. In summary, EP inhibited NSCLC cell growth, invasion and migration and induced apoptosis by suppressing the HMGB1/RAGE axis and the NF‑κB/STAT3 pathway, thus suggesting that EP may be a valuable therapeutic agent for NSCLC.