The role of high-mobility group box-1 (HMGB1) in outcome prediction in sepsis is controversial. Furthermore, its association with necroptosis, a programmed cell necrosis mechanism, is still unclear. The purpose of this study is to identify the association between the plasma levels of HMGB1 and the severity and clinical outcomes of sepsis, and to examine the correlation between HMGB1 and key executors of necroptosis including receptor-interacting kinase 3 (RIPK3) and mixed lineage kinase domain-like- (MLKL) proteins. Plasma HMGB1, RIPK3, and MLKL levels were measured with the enzyme-linked immunosorbent assay from the derivation cohort of 188 prospectively enrolled, critically-ill patients between April 2014 and December 2016, and from the validation cohort of 77 patients with sepsis between January 2017 and January 2019. In the derivation cohort, the plasma HMGB1 levels of the control (n = 46, 24.5%), sepsis (n = 58, 30.9%), and septic shock (n = 84, 44.7%) groups were significantly increased (P < 0.001). A difference in mortality between high (≥ 5.9 ng/mL) and low (< 5.9 ng/mL) HMGB1 levels was observed up to 90 days (Log-rank test, P = 0.009). There were positive linear correlations of plasma HMGB1 with RIPK3 (R2 = 0.61, P < 0.001) and MLKL (R2 = 0.7890, P < 0.001). The difference in mortality and correlation of HMGB1 levels with RIPK3 and MLKL were confirmed in the validation cohort. Plasma levels of HMGB1 were associated with the severity and mortality attributed to sepsis. They were correlated with RIPK3 and MLKL, thus suggesting an association of HMGB1 with necroptosis.