This study was carried out to determine the serum levels of high mobility group box protein 1 (HMGB1) in patients with ankylosing spondylitis (AS) and to evaluate its correlation with disease activity and quality of life. According to our knowledge, it is the first trial evaluating HMGB1 levels in AS. Serum samples of 30 patients (18 males and 12 females) with AS and 29 healthy controls (HC) (15 females and 14 males) were collected. HMGB1 levels were measured by enzyme-linked immunosorbent assay, activity of disease was assessed according to the Bath AS Disease Activity Index (BASDAI), and functional status of patients was evaluated with Bath AS Functional Index (BASFI). Modified Schober, chest expansion values and AS Quality of Life Questionnaire (ASQoL) scores were noted. The serum levels of HMGB1 were obtained significantly increased in AS patients compared to HC (p < 0.05). There was no significant correlation between HMGB1 levels and ESR (p > 0.05), and CRP (p > 0.05) values. BASDAI, BASFI and ASQoL scores were also not correlated with serum levels of HMGB1 (p > 0.05). Our results suggest that HMGB1 might play an important role in the pathogenesis of AS; however, it seems not to be a good candidate for reflecting disease activity, functional abilities and the quality of life in patients with AS; on the other hand, the increased levels of HMGB1 in patients may open a new dimension for targeting this cytokine as a new therapy option in AS.