Performance of two diagnoses, T-SPOT.TB (T-SPOT) and QuantiFERON-TB Gold (QFT-G), was compared in Japanese subjects. Forty-seven confirmed tuberculosis patients and eighty-four healthy subjects were recruited. All samples were assessed for both T-SPOT and QFT-G, and the sensitivities and the specificities were compared between two methods. The sensitivity was 100% for T-SPOT, and 87.2% for QFT-G. The specificity was 83.3 and 98.8%, respectively. The overall agreement of two tests was substantially high (Kappa coefficient = 0.671). The sensitivity of T-SPOT appeared to be higher than that of QFT-G, whereas the specificity of T-SPOT was significantly lower than that of QFT-G. The difference in the performance between T-SPOT and QFT-G and biological relevance of each system in diagnosing M. tuberculosis infection should be further explored.