High mobility group box 1 (HMGB1) has the capability of activating the immune response and taking part in macrophage polarization. Despite this, there is significant scope for exploration into how HMGB1 regulates macrophage polarization phenotype and influences intestinal epithelial barrier function. Investigating the role of HMGB1 in the creation of neutrophil extracellular traps (NETs) and the mechanism of its impact on macrophages could provide novel insights into intervening in intestinal inflammation and barrier damage. Therefore, the research examined the relationship between the macrophage polarization phenotype and HMGB1. Additionally, we analyzed how cell proliferation and cytokines changed in CaCo-2 cells following co-culture with HMGB1-influenced macrophages and intestinal epithelial CaCo-2 cells. We discovered that up-regulation of HMGB1 expression enhanced the creation of NETs, whereas inhibition of NETs formation led macrophages to switch from the anti-inflammatory M2 phenotype to the pro-inflammatory M1 phenotype. Additionally, we observed that macrophages induced by NETs containing HMGB1 can prompt CaCo-2 cell apoptosis and exacerbate the inflammatory response. HMGB1-containing NETs hinder tight junction protein expression in CaCo-2 cells by inducing macrophage M1 polarization, thereby impairing intestinal epithelial barrier function. Therefore, our findings indicate that by inhibiting the expression of HMGB1, the formation of NETs can be inhibited. This, in turn, mediates macrophage polarization and offers potential new therapies for intestinal diseases.