Gynecomastia (GYN) is the most common benign disease in males. A vacuum-assisted biopsy is a minimally invasive surgical technique for GYN treatment that achieves satisfactory aesthetic results. However, due to the operation under non-direct vision, it is difficult to localize the bleeding points and assess the residual glandular tissue. Endoscopy was applied to observe the operative field after subcutaneous mastectomy. The present study aimed to recommend our initial experience in glandular GYN with endoscope-assisted minimally invasive subcutaneous mastectomy. A total of 34 patients diagnosed with glandular GYN (50 breasts), treated with endoscope-assisted minimally invasive surgery at The First Affiliated Hospital with Nanjing Medical University between June 2018 and June 2020, were enrolled in this study. According to Simon's classification of the breast, 10 was grade I, 25 was grade IIA, and 15 was grade IIB. The characteristics of patients, operative data, postoperative complications, cosmetic outcome, and patient satisfaction were recorded. Endoscope-assisted minimally invasive mastectomy was performed successfully in all cases. The operative duration of the operation was 55-120min/side. The total weight of the resected tissue of the 50 breasts was 55-350g, and the blood loss was 10-105mL/breast. Endoscopy detected five breasts with bleeding and three with residual glandular during the operation. Postoperative bleeding occurred in 1 breast, subcutaneous seroma in 3 breasts, dysesthesia of the nipple-areolar complex in 2 breasts, and skin redundancy in a bilateral patient. None of the patients experienced severe pain, infection, nipple necrosis, and nipple retraction, a saucer-like deformity. With a median follow-up of 21months, all patients were satisfied with their cosmetic outcome (100%), and no recurrence occurred. Endoscope-assisted minimally invasive mastectomy could be used as a feasible technique for the treatment of glandular GYN. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online. Instructions to Authors www.springer.com/00266 .