Systemic brucellosis caused by Brucella melitensis biovar 3 was identified in a chamois (Rupicapra rupicapra) near the Parc National des Ecrins in the southern French Alps (France). Clinical signs included orchiepididymitis, polyarthritis, blindness and various neurological signs; necropsy findings included numerous calcified foci in testis, epididymis, kidney, subcutaneous connective tissue and brain. Brucella sp. were identified in brain by indirect immunofluorescence and B. melitensis biovar 3 was isolated from testis, kidney, eye, lung and joints. This report describes the first case of brucellosis and Brucella sp. isolation in chamois in France and the first case of B. melitensis isolation in this host species.