Ankylosing spondylitis (AS) is a chronic inflammatory disorder characterized by dysregulated T cells. We hypothesized that the aberrant expression of microRNAs (miRNAs) in T cells might involve in the pathogenesis of AS. The expression profile of 270 miRNAs in T cells from five AS patients and five healthy controls were analyzed by real-time PCR. After initial analysis, thirteen miRNAs were found potential differentially expression. After validation using 22 AS patients and 18 healthy controls, we confirmed that miR-16, miR-221 and let-7i were overexpressed in AS T cells and their expression levels were positively correlated with the Bath Ankylosing Spondylitis Radiology Index of lumbar spine in AS patients. In addition, the protein molecules regulated by miR-16, miR-221 and let-7i were measured by Western blotting. We found that the protein levels of Toll-like receptor 4 (TLR4), a target of let-7i, in T cells from AS patients were decreased. Lipopolysaccharide (LPS), a TLR4 agonist, inhibited interferon gamma (IFN-γ) secretion by anti-CD3+ anti-CD28 antibodies-stimulated T cells from healthy controls but not AS patients. After transfection of let-7i into normal T cells, LPS promoted IFN-γ production after stimulated by anti-CD3+anti-CD28 antibodies, but the transfection of let-7i does not have effects on IFN-γ production in AS T cells. In summary, we found that miR-16, miR-221, and let-7i were overexpressed in AS T cells and their expression levels were associated with radiographic change. In the functional study, the increased let-7i expression facilitated the Th1 (IFN-γ) immune response in AS T cells.