Tuberculosis has existed from early prehistoric days to modern times. The main causative agents of tuberculosis worldwide are Mycobacterium tuberculosis (M. tuberculosis) and M. bovis, along with M. africanum, M. cenettii and M. microti; these species make up the 'M. tuberculosis complex'. This worldwide infection has been of special interest to palaeopathologists due to its characteristic bone lesions as well as its great antiquity. Historically, tuberculosis has been recognised in Japan for more than a thousand years. However, the origin and early prevalence of tuberculosis remain unknown. In the present study, we present the earliest evidence of skeletal tuberculosis found in the Aneolithic Yayoi period in Japan (ca. 300 BC to AD 300). The skeletal remains showing typical pathological changes of spinal tuberculosis were dated to between 454 BC and AD 124 by dendrochronological methods using coburied arrow-shield board and house columns made of Japanese cedar. We discuss the early prevalence of this infectious disease and its influence on the population history of the Japanese from prehistoric to Aneolithic times.