We investigated the epidemiology of Trypanosoma pestanai infection in European badgers (Meles meles) from Wytham Woods (Oxfordshire, UK) to determine prevalence rates and to identify the arthropod vector responsible for transmission. A total of 245 badger blood samples was collected during September and November 2009 and examined by PCR using primers derived from the 18S rRNA of T. pestanai. The parasite was detected in blood from 31% of individuals tested. T. pestanai was isolated from primary cultures of Wytham badger peripheral blood mononuclear cells and propagated continually in vitro. This population was compared with cultures of two geographically distinct isolates of the parasite by amplified fragment length polymorphism (AFLP) and PCR analysis of 18S rDNA and ITS1 sequences. High levels of genotypic polymorphism were observed between the isolates. PCR analysis of badger fleas (Paraceras melis) collected from infected individuals at Wytham indicated the presence of T. pestanai and this was confirmed by examination of dissected specimens. Wet smears and Giemsa-stained preparations from dissected fleas revealed large numbers of trypanosome-like forms in the hindgut, some of which were undergoing binary fission. We conclude that P. melis is the primary vector of T. pestanai in European badgers.