Adenocarcinoma in the anal canal associated with an anal fistula is extremely rare, and in most cases its origin is difficult to ascertain because the primary sites have already been destroyed before any diagnosis of malignancy is able to be made. We report herein the case of a 62-year-old man found to have papillary adenocarcinoma with partial mucinous carcinoma associated with an anal fistula. The tumor was not exposed to the mucosal surface of the anal canal or rectum and an abdominoperineal resection was carried out. Macroscopic findings suggested that the tumor had developed from the anal fistula; however, the tumor showed a positive results when tested for O-acetylated sialic acids. This test also proved positive in the mucus of normal rectal mucosa, but not in the mucus of the anal glands. We speculated that the results of these tests may indicate that this tumor could have originated from the rectal mucosa, from where it migrated into the anal fistula.