Even though Entamoeba histolytica (Eh)-induced host pro-inflammatory responses play a critical role in disease, we know very little about the host factors that regulate this response. Direct contact between host cell and Eh signify the highest level of danger, and to eliminate this threat, the host immune system elicits an augmented immune response. To understand the mechanisms of this response, we investigated the induction and release of the endogenous alarmin molecule high-mobility group box 1 (HMGB1) that act as a pro-inflammatory cytokine and chemoattractant during Eh infection. Eh in contact with macrophage induced a dose- and time-dependent secretion of HMGB1 in the absence of cell death. Secretion of HMGB1 was facilitated by Eh surface Gal-lectin-activated phosphoinositide 3-kinase and nuclear factor-κB signaling and up-regulation of histone acetyltransferase activity to trigger acetylated HMGB1 translocation from the nucleus. Unlike lipopolysaccharide, Eh-induced HMGB1 release was independent of caspase-1-mediated inflammasome and gasdermin D pores. In vivo, Eh inoculation in specific pathogen-free but not germ-free mice was associated with high levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and keratinocyte-derived chemokine, which was suppressed with HMGB1 neutralization. This study reveals that Eh-induced active secretion of the HMGB1 plays a key role in shaping the pro-inflammatory landscape critical in innate host defense against amebiasis.