Background: Helicobacter pylori (HP) is identified as the most common gastrointestinal (GI) infection agent in the world. According to some findings shepherd's Helicobacterial infection is due to their contacts to sheep. Considering the controversy, the transmission of HP from the milk of small ruminates to humans and the possibility of the Zenotic nature of the disease has not proven yet. Thus, recognition of the routes of transmission of bacterium to human is vital. Materials and Methods : Using random clustering, 100 samples (81 sheep and 19 goat's samples) were taken from 20 villages of Mashhad suburb during two years. A questionnaire regarding the health status of the shepherds and their family from the point of view GI discomfort was taken. All taken samples were incubated on two specific HP media, HPSPA (Helicobacter pylori Special Peptone Agar) and Columbia Agar including antibiotics. Following centrifugation, DNA extraction was carried out on all precipitated samples. The specific Urease C gene of HP was traced through polymerase-chain reaction (PCR). Results: Considering the negative results of both PCR and isolation tests, neither culture media nor PCR could prove the existence of HP gene or the Urease C gene for the specific HP in samples. Information showed that 20 of shepherds and 25 of their families and also 10 of both are complaining of GI discomfort, without any clear relation to HP. Conclusion: The results showed that two incubation procedures could not detect HPÂ or its gene, Urease C. Probably, the reason could be due to some multifactorial agents, essential for the determination of the strategy of prevention and health. Possibility of transmission of the agent from the small ruminants and milk to humans needs further investigation