Aim Ankylosing spondylitis (AS) is a chronic inflammatory disease. However, the key inflammatory cytokines disrupted in this disease are not well defined. In this study, we performed protein array and multiple protein quantification to investigate the differentially expressed cytokines in plasma between AS patients and healthy subjects. Method In the discovery cohort, 5 AS patients who never underwent biologic therapy and 5 gender‐ and age‐matched healthy subjects were enrolled in the protein array analysis. Another 40 AS patients and 20 healthy participants were recruited in the validation stage. In addition, the messenger RNA and protein levels of osteogenesis‐related genes were quantified in hFOB1.19 cells in an in vitro osteoblast model. Results Of the 318 cytokines found to be differentially expressed by protein array, leukemia inhibitor factor (LIF) was significantly increased in AS patients as compared to controls. The “signaling by interleukins” pathway was the most enriched pathway in AS patients, and “signaling by interleukins”‐related cytokines, including LIF, interleukin (IL)‐6, IL‐23, and IL‐31, were significantly differentially expressed in the validation stage. Additionally, we correlated the expression of LIF with C‐reactive protein (CRP) and inflammation of magnetic resonance imaging lesions in the spine (MRI‐SPINE) in AS patients. We further analyzed the effects of LIF in hFOB cells and found that LIF promoted the growth factor receptor‐bound protein 2 / phospho‐extracellular signal‐regulated kinase / runt‐related transcription factor 2 / alkaline phosphatase pathway at the protein level and activated several osteogenesis‐related genes (RUNX2 and BGLAP). Conclusion LIF was increased in the plasma of AS patients as compared with healthy subjects and significantly correlated with inflammation indices (CRP and MRI‐SPINE) in AS patients. Thus, LIF may play a critical role in the pathogenesis of AS via promoting osteogenic differentiation.