The nuclear protein high-mobility group box 1 (HMGB1) can be passively released by necrotic cells or secreted actively by several cell types to regulate immune and inflammatory responses, as well as tissue remodeling. We herein aimed to characterize the effect of insulin resistance on HMGB1 in adipose tissue and to examine its potential role as a metabolic regulator in β-pancreatic cells. Plasma HMGB1 concentration and adipose HMGB1 expression were assessed in relation to obesity and insulin resistance. Cultured adipocytes from lean and obese patients were used to investigate the intracellular distribution and factors regulating HMGB1 release, as well as to test its effects on adipogenesis and lipid metabolism. A regulatory role for HMGB1 in insulin secretion was also investigated. Circulating HMGB1 was positively associated with body mass index, while adipose HMGB1 mRNA levels correlated with the expression of inflammatory markers. Insulin resistance modified the intracellular distribution of HMGB1 in human adipocytes, with HMGB1 being predominantly nuclear in lean and obese normoglycemic individuals while localized to the cytosol in obese patients with type 2 diabetes. Adipocytes from lean individuals exposed to conditioned media from lipopolysaccharide-stimulated macrophages induced HMGB1 redistribution to the cytoplasm and release. HMGB1 treatment had no effect on differentiation and lipid metabolism in adipocytes. However, HMGB1, whose circulating levels correlated with postload insulin concentration, increased both insulin release and intracellular Ca(2+) concentration in INS-1 cells. These findings show, for the first time, that HMGB1 expression and release by human adipocytes is altered by inflammatory conditions as those imposed by obesity and insulin resistance. Our data reveal a novel role for HMGB1 as a stimulatory factor of insulin secretion of β-pancreatic cells.