Abstract. The aim of the present study was to evaluate the association between follicular fluid (FF) levels of high-mobility group box 1 (HMGB1) protein and the reproductive outcome in patients undergoing in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). FF samples were collected from the ovarian follicles (≥14 mm) of 143 infertile patients that had undergone IVF/ICSI, and the HMGB1 expression levels were determined using ELISA. Spearman's correlation and receiver operating characteristic (ROC) curve analysis were applied to analyze the results. Significantly increased levels of HMGB1 protein (7.38±2.02 vs. 6.14±2.52 ng/ml; P<0.01), endometrial thickness on the day of human chorionic gonadotropin (hCG) administration (10.3±1.3 vs. 9.7±1.7 mm; P<0.01) and retrieved oocyte counts (11.68±6.51 vs. 11.00±6.34; P<0.01) were observed in the pregnant group when compared with the non-pregnant group. Conversely, the level of luteinizing hormone on the day of hCG administration was significantly reduced in the pregnant group compared with the non-pregnant group (0.92±1.78 vs. 1.78±2.03 pmol/l, P<0.01). The ROC curve indicated a significant association between the FF level of HMGB1 protein and the pregnancy rate, with an area under the ROC curve of 0.673 (0.581-0.765; P<0.01). In addition, the HMGB1 protein level was shown to have a significant positive correlation with the endometrial thickness (r=0.170; P<0.05). Therefore, the present study indicated that the FF levels of HMGB1 protein are increased in pregnant patients and are positively correlated with endometrial thickness. Thus, FF levels of HMGB1 may be a useful factor for predicting the outcome of IVF/ICSI treatments.