Ankylosing spondylitis (AS) is a chronic inflammatory disease mainly affecting the spine and sacroiliac joints. Mediators such as macrophage migration inhibitory factor (MIF) and interleukin-10 (IL-10) are thought to be involved in several inflammatory conditions, including AS. Proinflammatory cytokines regulate the production of oxidative stress markers, such as nitric oxide (NO) and malondialdehyde (MDA). Although oxidative stress and lipid peroxidation have been reported in AS, the association of AS with commonly known oxidative stress markers and cytokines remains uncertain. We have therefore studied whether serum MIF levels are elevated in patients with AS and whether the levels correlate with oxidative stress markers and disease activity parameters. Twenty-five AS patients and 18 healthy controls participated in this study; subjects with hypertension, diabetes, hyperlipidemia, and obesity were excluded. The levels of acute phase reactants, serum levels of glucose, lipids, MIF, IL-10, NO and MDA were studied. Spinal mobility was assessed by the Bath Ankylosing Spondylitis Metrology Index (BASMI). Patients were also assessed using with the Bath Ankylosing Spondylitis Functional Index and the Bath Ankylosing Spondylitis Disease Activity Index. Age and sex distribution were found to be comparable between AS patients and controls (p > 0.05). Acute phase reactants and MIF levels were significantly higher (p < 0.05) and IL-10 levels were significantly lower (<0.001) in the AS patients than in controls. There was a significant correlation between BASMI and MIF levels in AS patients (r = 0.714, p < 0.001). Based on these results, MIF may be involved in the pathogenesis of the chronic inflammation in AS and, consequently, targeting MIF may be beneficial in preventing complications or in initiating early treatment of the disease.