Background. The spirochete Borrelia miyamotoi belongs to the group of borrelia causing tick-borne relapsing fevers. It is transmitted by the same tick species as Borrelia burgdorferi sensu lato and is widespread in temperate climate zones of Eurasia and North America. Previously we have shown by PCR that clinical cases presented as systemic febrile illness and diagnosed usually as «Ixodes tick-borne borreliosis without erythema migrans» were caused by B. miyamotoi. According to recent estimation in Russia there are several thousands human cases of B. miyamotoi infection annually. The investigation of this emerging human pathogen was hampered because the attempts to culture this bacterium directly from human blood failed. Results and conclusion: We here describe protocol - avoiding the inhibitory effects of anticoagulants on B. miyamotoi growth - enabling successful B. miyamotoi isolation from blood of Russian patients.