This study aims to investigate the relationships between the serum concentrations of High Mobility Group Box 1 (HMGB1) protein and Tumor Necrosis Factor-α (TNF-α), coronary artery lesions, and cardiac function in patients with Acute Coronary Syndrome (ACS). We prospectively followed healthy controls (n=30) and patients with ACS (n=60) who underwent coronary angiography. Serum HMGB1 and TNF-α concentrations were measured by enzyme-linked immunosorbent assay. Serum concentrations of HMGB1 (33.22 ± 16.59 ng/ml in Acute Myocardial Infarction Group (AMIG) and 23.40 ± 12.17 ng/ml in Unstable Angina Pectoris Group (UAPG)) and TNF-α (10.68 ± 4.94 pg/ml in AMIG and 6.37 ± 3.53 pg/ml in UAPG) in patients with ACS prior to standard hospitalization were significantly higher than those of the control group (HMGB1: 12.23 ± 2.74 ng/ml; TNF-α: 2.13 ± 1.26 pg/ml) (p<0.01). The concentrations of HMGB1 (21.67 ± 13.00 ng/ml in AMIG and 18.71 ± 6.92 ng/ml in UAPG) and TNF-α (5.28 ± 3.09 pg/ml in AMIG and 3.71 ± 2.34 pg/ml in UAPG) in patients with ACS post-standardized hospitalization were lower than those prior to hospitalization (p<0.05). Correlation analysis revealed a positive correlation between the serum concentration of HMGB1 and coronary artery lesion counts and severity as evaluated using the Gensini integral (r=0.525, p<0.01 and r=0.588, p<0.01, respectively), and a negative correlation with left ventricular ejection fraction (r=-0.488, p<0.01) in patients with ACS. Moreover, the serum concentration of TNF-α was positively correlated with that of HMGB1 (r=0.415, p<0.01). The serum concentrations of HMGB1 and TNF-α may be useful indicators to estimate the severity of coronary artery lesions in patients with ACS.