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The aim of this study was to explore a technique that uses real-time fusion image of single-photon emission computed tomography/computed tomography (SPECT/CT) to guide percutaneous transthoracic needle biopsy (PTNB) and test its feasibility and safety in patients. Twelve patients with intrathoracic lesions suspicious for malignancy underwent real-time 99m-technetium sestamibi SPECT/CT-guided PTNB. Chest SPECT/CT fusion images were used for biopsy planning and intraprocedure fusion of SPECT with guiding CT was used to adjust the puncture angle and direction. Tissues were sampled when the tip of needle was placed on the margin of the hyperactive area within the mass. Postoperation CT scan was performed to exclude acute complications. The technical success rate, complications, and histological results were recorded. Room time and operation time were calculated. All biopsy procedures fulfilled the primary endpoint of yielding diagnostic results: lung adenocarcinoma (n=5), lung squamous cell carcinoma (n=4), lung small cell carcinoma (n=1), mediastinum lymphoma (n=1), and pleura inflammation (n=1). There were no major complications. The mean room time was 74.3±10.4 (range: 57–91) min, and the mean operation time was 33.5±9.1 (range: 15–45) min. The average number of CT guiding scans was 3.2 (range: 1–4). The pilot study explored a new technique of real-time guidance of PTNB using SPECT/CT and verified its feasibility and safety.