Fecal samples from 444 Danish patients presenting with acute diarrhea were tested for Blastocystis and positive samples were subtyped to investigate the prevalence and subtype distribution of Blastocystis in this patient group. A total of 25 patients (5.6%) were positive, and 19 of these patients (76.0%) were positive for Blastocystis sp. ST4. Because the relative prevalence of ST4 in other patients presenting with other types of diarrhea (persistent, travel-related, and human immunodeficiency virus-related) in Denmark is low, the role of Blastocystis sp. ST4 in the etiology of acute diarrhea should be investigated further.