Introduction Ankylosing spondylitis (AS) and inflammatory bowel disease (IBD) share similarities and are classified as spondyloarthropathies. In IBD, anti-Saccharomyces cerevisiae antibody (ASCA), anti-I2 (associated with anti-Pseudomonas activity), anti-Escherichia coli outer membrane porin C (anti-OmpC), anti-flagellin (anti-CBir1), and antineutrophil cytoplasmic antibodies (ANCA) possess clinical significance. Because of the overlap between the two conditions, a pilot study was designed to compare the frequency of these antibodies in AS patients compared to normal controls. Methods Serum stored from 80 AS patients and 80 control subjects was available for analysis. ASCA, anti-I2, anti-OmpC, anti-CBir1, and ANCA studies were completed on all serum samples using Enzyme-Linked Immunosorbent Assay (ELISA) methodology. The following analyses were performed: comparison of positivity based on the established values in IBD, median values, the number of subjects in each serology in the 4th quartile of a normal distribution, and the mean quartile sum of all the antibodies. Results There was no difference in positivity rates between AS and control groups with the established IBD values. The median anti-I2 response was significantly higher in AS than in controls (11.78 vs 7.86, p = 0.017). Significantly more AS patients had quartile scores of 4 for the following antibody responses: ASCA IgG (26% vs 13%, p = 0.016, OR = 2.49, CI 1.168 - 5.313), ASCA IgG and IgA (27% vs 12%, p = 0.006, OR = 2.9, CI: 1.342 - 6.264), and anti - I2 (25% vs 14%, p = 0.0424, OR = 2.15, CI: 1.018 - 4.538). The mean quartile sum of the antibody responses was elevated in AS patients when ANCA was excluded (10.526 vs 9.519, p = 0.03). When ANCA was included, this difference lost significance. Conclusions The data from this pilot study points towards mucosal dysregulation as an important pathway in AS. We were able to demonstrate that anti-I2 could play a pathologic role in AS. The elevated mean total antibody response being significant only with ANCA exclusion is consistent with the histopathological evidence that intestinal inflammation in AS is similar to Crohn's disease. To better define the roles of these antibodies in AS, larger studies with more precisely defined patient characteristics are required.