This study aimed to examine the diagnostic accuracy and clinical efficacy of initial CT-guided percutaneous biopsy of the vertebral lesions. A total of 305 percutaneous biopsies of the vertebral lesions were performed under either CT guidance (n=127) or C-arm guidance (n=178). The diagnostic accuracy rate was evaluated by comparing the histopathological diagnosis with the ultimate diagnosis. The histopathological diagnosis was consistent with the ultimate diagnosis in 108 (85.0%, 108/127) cases of CT-guided biopsy and in 135 (75.8%, 135/178) cases of C-arm guided biopsy and there was a significant difference. The accuracy of diagnosis based on biopsies varied with different diseases, including primary benign or malignant tumors, metastatic tumors, inflammatory lesions and fractures. A second biopsy or further examinations were required for patients with negative result obtained in the initial biopsy. The complication rate was 3.1% (4/127) in CT-guided biopsy and 7.3% (13/178) in C-arm guided biopsy. In conclusion, CT-guided percutaneous biopsy is an accurate and safe technique for biopsy of the vertebral lesions.