Blastocystis has a widespread distribution in a variety of animals, which is a potential source of infection for humans. However, the contribution of zoonotic transmission remains unclear due to the absence of molecular proof of these organisms being identical to those found in humans. We report herein the similar subgroup of Blastocystis isolates from humans, pigs, and a horse using a restriction fragment length polymorphism (RFLP) analysis of partial small-subunit ribosomal DNA (ssu rDNA). Additionally, sequence and phylogenic analysis of partial ssu rDNA of Blastocystis from a human, a pig, and a horse sharing a common subgroup shows that Blastocystis isolates from a pig and a horse were monophyletic and closely related to B. hominis, with 92 to 94% identity. These results suggest the possibility of zoonotic potential of Blastocystis.