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Background To analyze the feasibility of active breath control (ABC), the lung tumor reproducibility and the rationale for single-breath-hold cone beam CT (CBCT)-guided hypofraction radiotherapy. Methods Single-breath-hold CBCT images were acquired using ABC in a cohort of 83 lung cancer patients (95 tumors) treated with hypofraction radiotherapy. For all alignments between the reference CT and CBCT images (including the pre-correction, post-correction and posttreatment CBCT images), the tumor reproducibility was evaluated via online manual alignment of the tumors, and the vertebral bone uncertainties were evaluated via offline manual alignment of the vertebral bones. The difference between the tumor reproducibility and the vertebral bone uncertainty represents the change in the tumor position relative to the vertebral bone. The relative tumor positions along the coronal, sagittal and transverse axes were measured based on the reference CT image. The correlations between the vertebral bone uncertainty, the relative tumor position, the total treatment time and the tumor reproducibility were evaluated using the Pearson correlations. Results Pre-correction, the systematic/random errors of tumor reproducibility were 4.5/2.6 (mediallateral, ML), 5.1/4.8 (cranial-caudal, CC) and 4.0/3.6 mm (anterior-posterior, AP). These errors were significantly decreased to within 3 mm, both post-correction and post-treatment. The corresponding PTV margins were 4.7 (ML), 7.4 (CC) and 5.4 (AP) mm. The changes in the tumor position relative to the vertebral bone displayed systematic/random errors of 2.2/2.0 (ML), 4.1/4.4 (CC) and 3.1/3.3 (AP) mm. The uncertainty of the vertebral bone significantly correlated to the reproducibility of the tumor position (P < 0.05), except in the CC direction post-treatment. However, no significant correlation was detected between the relative tumor position, the total treatment time and the tumor reproducibility (P > 0.05). Conclusions Using ABC for single-breath-hold CBCT guidance is an effective method to reduce the PTV margin of hypofraction radiotherapy for lung cancer. Using ABC, the tumor position was significantly altered relative to the vertebral position. The reproducibility of the tumor position was affected by the vertebral bone but not by the relative tumor position or the total treatment time. [ABSTRACT FROM AUTHOR]