Ankylosing spondylitis (AS) is a chronic, disabling rheumatic disease with indefinite etiology. Human leukocyte antigen-B27 (HLA-B27) carriage and Klebsiella pneumoniae (K. pneumoniae) infections may contribute to the etiopathogenesis of AS. The objective of this study was to determine the association of HLA-B27 carriage and serum immunoglobulin G (IgG) to K. pneumoniae with AS and its clinical outcome. In a case-control study, HLA-B27 carriage was detected by polymerase chain reaction, serum IgG to K. pneumoniae was measured by ELISA, and K. pneumoniae was isolated from the stool of 40 AS patients who were compared to 40 age- and sex-matched normal individuals. Clinical findings, disease activity, and functional ability were evaluated for all AS patients. HLA-B27, serum IgG to K. pneumoniae, and fecal carriage of Klebsiella were significantly higher in AS patients when compared to controls (p < 0.001 for all). Disease activity and functional score categories were significantly higher in HLA-B27 positive AS patients with an elevated titer of K. pneumoniae IgG than in HLA-B27 negative patients with a low titer of K. pneumoniae IgG (p < 0.012 and p < 0.001, respectively). In conclusion, HLA-B27 carriage and K. pneumoniae infections could play a significant role in the development and clinical outcome of AS patients.