Blastocystis has a widespread distribution in a variety of animals, which is a potential source of infection for humans. Previous studies show that Blastocystis sp. subtypes 1-4, 6, and 7 were composed of isolates from humans and animals, while Blastocystis sp. subtype 5 included only pig and cattle isolates. A more recent study on the basis of the SSU rDNA sequence has showed that a single Blastocystis isolate amplified directly from the faeces of a Thai human belongs to Blastocystis sp. subtype 5, but that study failed to cultivate this isolate. We report herein two human isolates from in vitro cultures belonging to Blastocystis sp. subtype 5 and one human isolate from in vitro culture containing two distinct genotypes of Blastocystis sp. subtypes 3 and 5 using PCR amplification with seven kinds of sequence-tagged site (STS) primers. Additionally, 16 Blastocystis isolates from pigs living in the same rural area with the three humans infected Blastocystis sp. subtype 5 were also genotyped by PCR with the STS primers, and all isolates from pigs and humans were compared by small-subunit ribosomal RNA (SSU rRNA) restriction-fragment-length polymorphism (RFLP) analyses using two restriction endonucleases (HinfI and RsaI). The results indicated that all of the isolates from pigs showed Blastocystis sp. subtype 5 and the RFLP patterns of all of the isolates from humans except for the mixed one were identical or quite similar to those of the 16 pig isolates with both HinfI and RsaI enzymes. These findings provide additional molecular-based evidence supporting the zoonotic potential of Blastocystis sp. subtype 5. This study also showed that Blastocystis sp. subtype 3 overgrew Blastocystis sp. subtype 5 in vitro.