Encephalitozoon cuniculi is an obligate intracellular microsporidian that is the causal agent of encephalitozoonosis, an important and emerging disease in both humans and animals. Little is known about its occurrence in wildlife. In this study, serum samples from 793 wild rodents [178 bank voles (BV), 312 field voles (FV) and 303 wood mice (WM)], 96 foxes and 27 domestic cats from three study areas in the UK were tested for the presence of antibodies to E. cuniculi using a direct agglutination test (DAT). Seroprevalence in the wild rodents ranged from 1.00% to 10.67% depending on species (overall 5.31%) and was significantly higher in foxes [49.50% (50/96)]. None of the 27 cats sampled were found to be seropositive. This is the first report of seroprevalence to E. cuniculi in BV, FV, WM, foxes and cats in the UK and provides some evidence that foxes could act as sentinels for the presence of E. cuniculi in rodents. The study demonstrates that wildlife species could be significant reservoirs of infection for both domestic animals and humans.