Abstract Background Ankylosing spondylitis (AS) shares many characteristics with inflammatory bowel disease (IBD). Intestinal microbiota most likely plays an important role in the development of IBDs and may also be involved in the pathogenesis of AS. We aimed to define and compare the fecal microbiota composition in patients with AS, ulcerative colitis (UC), and healthy controls (HC) and to determine relationships between fecal microbiota, fecal calprotectin, and disease-related variables in AS. Methods Fecal microbiota composition was assessed with GA-map™ Dysbiosis Test (Genetic Analysis, Oslo, Norway), which also reports the degree of deviation of the microbiota composition compared with a healthy control population, a Dysbiosis Index (DI) score 1–5. The AS patients were assessed with questionnaires, back mobility tests, fecal calprotectin, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). Results Totally, 150 patients with AS (55% men, median age 55.5 years, median BASDAI 3.2), 18 patients with UC (56% men, median age 30.5 years), and 17 HC (65% men, median age 22 years) were included. Principal component analysis showed highly separate clustering of fecal microbiota from the patients with AS, UC, and HC. Compared with HC, fecal microbiota in AS was characterized by a higher abundance of Proteobacteria, Enterobacteriaceae, Bacilli, Streptococcus species, and Actinobacteria, but lower abundance of Bacteroides and Lachnospiraceae. Further, fecal microbiota composition differed between patients with normal (≤ 50 mg/kg, n = 57) and increased (≥ 200 mg/kg, n = 36) fecal calprotectin. Patients with increased fecal calprotectin had lower abundance of bacteria with anti-inflammatory properties such as Faecalibacterium prausnitzii and Clostridium and higher abundance of the genus Streptococcus. No association was found between the fecal microbiota composition and HLAB27 status, disease activity, function, or medication. Dysbiosis (defined as DI ≥ 3) was found in 87% of AS patients. Conclusions Patients with AS have a distinct fecal microbiota signature, which is linked to fecal calprotectin levels, a marker of intestinal inflammation, but not to other clinical parameters. These findings suggest a local interplay between intestinal microbiota and gut inflammation in AS. Trial registration ClinicalTrials.gov, NCT00858819. Registered March 9, 2009.