High mobility group box B1 (HMGB1)-receptor for advanced glycation endproducts (RAGE) axis has been previously known to be involved in carcinogenesis and development of multiple malignancies. Some studies have confirmed that Ethyl pyruvate (EP), a potent inhibitor of HMGB1, exerts the therapeutic effects on metastatic live tumor from gastric cancer. However, the effects and possible molecular mechanisms of EP on gallbladder cancer (GBC) need to be further explored. In the present study, human GBC cell lines (GBC-SD and SGC-996) were treated with different concentrations of EP. Then, the expression levels of HMGB1, RAGE and some transcription factors were identified by Real-time PCR and Western blot assays. Cell proliferative activities indicated by MTT assay, invasive potential by Transwell assay and cell apoptosis and cycle distribution were performed for functional analysis of GBC cell lines in vitro. As a result, EP decreased the expression of HMGB11, RAGE, PCNA and matrix metallopeptidase-9 (MMP-9), while it increased the expression of p53. Moreover, EP administration decreased GBC cell proliferation, inhibited the invasive potential, and induced apoptosis and cycle arrest in S phase in GBC cells. In conclusion, EP administration inhibits growth and invasion of gallbladder cancer cells possibly via down-regulation of the HMGB1-RAGE axis, suggesting that EP may play a critical role in the treatment of cancer in conjunction with other therapeutic agents. Copyright © by BIOLIFE, s.a.s.