Objective: To investigate the predictive value of serum high-mobility group box-1 protein (HMGB1) for hemorrhage transformation (HT) after intravenous thrombolysis in patients with acute ischemic stroke. Methods: From February 2017 to September 2019, patients with acute ischemic stroke underwent intravenous thrombolysis in Lixin County People's Hospital, Bozhou, Anhui Province were enrolled prospectively. In the morning of the day after admission, fasting blood was collected to detect the level of serum HMGB1. Twenty-four hours after intravenous thrombolysis, CT reexamination was performed to determine whether HT occurred. The demographic and baseline clinical data were compared between the HT group and the non-HT group. Multivariate logistic regression analysis was used to determine the independent risk factors for HT after thrombolysis. Receiver operating characteristic (ROC) curve was used to analyze the predictive value of serum HMGB1 level to HT. Results: A total of 182 patients were enrolled in the study, including 22 in the HT group and 160 in the non-HT group. The age, fasting blood glucose, serum HMGB1 level, and the proportion of history of atrial fibrillation and regular antiplatelet medication before onset in the HT group was significantly higher than those in the non-HT group, and the differences were statistically significant (all P<0.05). Multivariate logistic regression analysis showed that the increased serum HGMB1 level (odds ratio [OR] 2.145, 95% confidence interval[CI] 1.467-3.138; P=0.002), taking antiplatelet drugs regularly before onset (OR 5.496, 95% CI 1.700-17.768; P=0.004) and increased baseline fasting blood glucose level (OR 1.333, 95% CI 1.024-1.736; P=0.033) were the independent risk factors for HT after intravenous thrombolysis. ROC curve analysis showed that the area under the curve of serum HMGB1 level predicting HT after intravenous thrombolysis was 0.788 (95% CI 0.721-0.845; P<0.001). The sensitivity and specificity were 72.73% and 82.50%, respectively, when the best cutoff value was 7.97 μg/L. Conclusion The increased baseline HMGB1 level may predict the risk of HT after intravenous thrombolysis in patients with acute ischemic stroke.