The purpose of this study was to explore the feasibility of subtraction coronary computed tomography angiography (CCTA) by second-generation 320-detector row CT in patients with severe coronary artery calcification using invasive coronary angiography (ICA) as the gold standard. This study was approved by the institutional board, and all subjects provided written consent. Twenty patients with calcium scores of >400 underwent conventional CCTA and subtraction CCTA followed by ICA. A total of 82 segments were evaluated for image quality using a 4-point scale and the presence of significant (>50 %) luminal stenosis by two independent readers. The average image quality was 2.3 ± 0.8 with conventional CCTA and 3.2 ± 0.6 with subtraction CCTA (P < 0.001). The percentage of segments with non-diagnostic image quality was 43.9 % on conventional CCTA versus 8.5 % on subtraction CCTA (P = 0.004). The segment-based diagnostic accuracy for detecting significant stenosis according to ICA revealed an area under the receiver operating characteristics curve of 0.824 (95 % confidence interval [CI], 0.750-0.899) for conventional CCTA and 0.936 (95 % CI 0.889-0.936) for subtraction CCTA (P = 0.001). The sensitivity, specificity, positive predictive value, and negative predictive value for conventional CCTA were 88.2, 62.5, 62.5, and 88.2 %, respectively, and for subtraction CCTA they were 94.1, 85.4, 82.1, and 95.3 %, respectively. As compared to conventional, subtraction CCTA using a second-generation 320-detector row CT showed improvement in diagnostic accuracy at segment base analysis in patients with severe calcifications.