Background: Metastasis to the adrenal glands is frequent in patients with various cancers and adrenal gland biopsy is routinely performed using ultrasound or computed tomographic (CT) guidance. However, this method is technically challenging, especially in the case of small masses. Purpose: To determine whether the new real-time stereotactic needle guidance technique C-arm cone-beam CT (CBCT) allows safe and accurate biopsy of adrenal gland masses, especially those in hard-to-reach anatomical locations. Material and Methods: CBCT guidance was used to perform 60 stereotactic biopsy procedures of lesions that were inaccessible with ultrasound or CT guidance. The needle path was carefully planned and calculated on the CBCT virtual navigation guidance system, which acquired 3D CT-like cross-sectional images. The adrenal biopsy procedures were performed with fluoroscopic feedback. Technical success rate, sensitivity, specificity, accuracy, and complications were investigated. Results: The technical success rate of adrenal biopsy under CBCT virtual navigation was 100%, with a mean total procedure time of 14.6 +/- 3.6min. Of the 60 lesions, 46 were malignant, 11 were benign, and three were non-diagnostic. The three non-diagnostic lesions proved to be malignant. Thus, the sensitivity, specificity, and accuracy were 93.8%, 100%, and 95.0%, respectively. Minor bleeding occurred in two (3.3%) cases. Conclusion: CBCT guidance allows safe and accurate biopsy of adrenal gland masses and may be especially useful for hard-to-reach anatomical locations.