Joint disease ankylosing enthesopathy (ANKENT) naturally occurs in inbred mice with C57Bl/10 genetic background. ANKENT has many parallels to human ankylosing spondylitis (AS) and represents an animal model for AS. Environmental conditions (i.e., microbial load of the organism) are among the risk factors for ANKENT, similar to AS. The role of microflora in the development of ANKENT was investigated. ANKENT was tested in four experimental groups of germ-free mice associated with different numbers of various intestinal microbes and three control groups: germ-free, specific pathogen-free, and conventional (CV) mice. Mice were colonized either with anaerobic bacteria isolated from the intestine of a CV mouse or with bacterial strains obtained from the collection of microorganisms. Microbes were characterized and checked by microbiological cultivation methods and with the use of polymerase chain reaction amplification and rDNA sequence analysis. Joint disease developed in GF mice colonized with a mixture containing Bacteroides spp. and Enterococcus sp., and/or Veillonella sp. and Staphylococcus sp. No ANKENT appeared in males colonized with probiotic bacterium Lactobacillus sp. In control groups ANKENT occurred in SPF and CV animals; the GF animals remained healthy. The results confirmed that the germ-free conditions protect from joint inflammation, and thus microbes are necessary for ANKENT development. In colonized mice the ANKENT was triggered by luminal anaerobic bacteria, which are common components of intestinal microflora.