Interleukin-33 (IL-33) is implicated in rheumatoid arthritis with effects of promoting tumor necrosis factor-alpha (TNF-α) and interleukin-17 (IL-17) productions, which have been demonstrated to play a pivotal role in ankylosing spondylitis (AS). However, changes of IL-33 levels and its effects in AS have not been investigated. Eighty-nine and 178 healthy controls were included in the current study. Erythrocyte sedimentation rate, serum levels of C-reactive protein, IL-17, and IL-33 were determined. Effects of IL-33 on TNF-α and IL-6 productions were investigated. Effects of IL-33 on neutrophil migration were also evaluated. Serum levels of IL-33 were elevated in AS patients. Moreover, IL-33 was significantly higher in active AS patients according to Bath Ankylosing Spondylitis Disease Activity Index. IL-33 concentrations in serum were positively correlated with TNF-α and IL-17 levels (IL-33 and TNF-α, r = 0.54, P < 0.01; IL-33 and IL-17, r = 0.47, P < 0.01). IL-33 dose-dependently enhanced TNF-α and IL-6 productions by peripheral blood mononuclear cells (PBMCs) responding to lipopolysaccharide. IL-33 induced neutrophil migration only in higher doses (≥10 ng/ml). Serum levels of IL-33 were elevated in AS patients. IL-33 may play a role in AS development via enhancing TNF-α production by PBMCs and inducing neutrophil migration.