Previously, we demonstrated that Agrocybe aegerita lectin (AAL), a galectin isolated from edible mushroom Agrocybe aegerita, exerts potent anti-tumor activity, while the mechanisms by which AAL suppresses tumor growth are yet to be elucidated. Here, we conducted studies with focus for its impact on the cecal ligation and puncture (CLP)-induced innate immune response. Administration of AAL significantly exacerbated the severity of CLP-induced septic shock as manifested the increased lethality. AAL promoted inflammatory cytokine production by preferentially regulating macrophage activation and recruitment. Mechanistic studies revealed that AAL likely targets macrophages through receptor Mincle to activate Syk/Card9 signaling, which then couples to the Nlrp3 inflammasome assembly. It was further noted that AAL markedly promotes H3K4 di- and trimethylation, by which it enhances Hmgb1 expression. Specifically, AAL induced macrophages secretion of copious amount of Hmgb1 as manifested the Hmgb1 cytoplasmic translocation along with the detection of extracellular Hmgb1. AAL also stimulated a significant increase for nuclear Hmgb1, which then formed a complex with RelA, and thereby enhancing NF kappa B transcriptional activity. Together, our data suggest that AAL may possess important pharmaceutical properties in the regulation of innate immune response.