Lyme borreliosis is a common tick-borne disease with a wide variety of clinical manifestations. Cardiac involvement has been reported during both the acute phase (atrioventricular block, pericarditis) and the chronic stage (dilated cardiomyopathy), but is rare (<5%). Here we describe the first case of Borrelia afzelii Lyme endocarditis, in a 61-year-old man living in an endemic area of France. The diagnosis was confirmed by detection of B. afzelii DNA in the mitral valve by specific real-time PCR. He was treated empirically with amoxicillin for 6 weeks and remains well 12 months later.