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Dientamoeba fragilis (D. fragilis) is a worldwide distributed protozoan parasite; it is pathogenic for humans. A wide spectrum of gastrointestinal symptoms has been described in infected patients: diarrhoea, flatulence, abdominal pains, colic and weight loss. However, asymptomatic infection has been also described. D. fragilis is still not well known; no cystic stage has been demonstrated and only the trophozoites are detected in stool samples. For identifying this typically more often binucleate protozoan, is necessary to perform permanent stain (eg. Giemsa) on fresh stool specimens. This protozoan is extremely difficult to cultivate but molecular techniques such as the Polymerase Chain Reaction offer promise as a means of diagnosing infection. In five years time (2006-2010), faecal samples were collected from pigs housed in farrow-to-finish herds (494 samples, splitted in three different categories: sows, growers, finishing pigs) and from hunted or slaughtered wild boars (87 samples). Simultaneously, the study was undertaken on human faeces (17 samples) to evaluate the presence of D. fragilis in pig breeders. All samples were collected directly from the rectum, cooled and sent to the laboratory where they were examined for D. fragilis by direct microscopic examination. The fresh faecal smears were stained with a 10% Giemsa solution in distilled water for 30 min. Biomolecular investigations (TaqMan real-time PCR which targets the 5.8S rRNA, nested PCR for the 18S rRNA, nested PCR for the internal transcribed spacer 1 region) were carried out on 38 pigs and 17 pig breeders specimens. The microscopic examination of the fresh fecal smears revealed positivity in 277 domestic pigs, corresponding to 56.07%. In particular higher positivity was observed on youngest animal (76.57%), while oldest or mature pigs recorded an important decreasing of positivity according the age (Table 1). Concerning wild boars, we revealed positivity in 35 animals (40.22%). Among humans, the positivity was 76.47% and these positive specimens came from people with a close contact with pigs. Biomolecular investigations carried out on human and animals amplified positive products revealed 100% homology with the 5.8S rRNA gene of D. fragilis, genotype 1 (e.g., Genbank DQ233451). During a five years research project we demonstrated the presence of D. fragilis in domestic pigs populations as well as in hunted or slaughtered wild boars. Due to the high percentage of positivity we could assume the domestic and/or wild pigs can play a role as natural reservoir of the parasite. In this scenario, outdoor pig farms and/or “confined” wild boars rearing can act as important link of exchange of this parasite. The demonstrated homology of D. fragilis sequences obtained from both humans and animals suggests the potential role of this parasite as zoonotic agent. If an environmentally resistant and infective stage of D. fragilis exists, we suppose the environmental contamination with domestic/wild pigs feces could be as an important factor in the transmission of this parasite to other hosts, including humans.