We used 3D printed-breast surgical guides (3DP-BSG) to designate the original tumor area from the pre-treatment magnetic resonance imaging (MRI) during breast-conserving surgery (BCS) in breast cancer patients who received neoadjuvant systemic therapy (NST). Targeting the original tumor area in such patients using conventional localization techniques is difficult. For precise BCS, a method that marks the tumor area found on MRI directly to the breast is needed. In this prospective study, patients were enrolled for BCS after receiving NST. Partial resection was performed using a prone/supine MRI-based 3DP-BSG. Frozen biopsies were analyzed to confirm clear tumor margins. The tumor characteristics, pathologic results, resection margins, and the distance between the tumor and margin were analyzed. Thirty-nine patients were enrolled with 3DP-BSG for BCS. The median nearest distance between the tumor and the resection margin was 3.9 cm (range 1.2-7.8 cm). Frozen sections showed positive margins in 4/39 (10.3%) patients. Three had invasive cancers, and one had carcinoma in situ; all underwent additional resection. Final pathology revealed clear margins. After 3-year surveillance, 3/39 patients had recurrent breast cancer. With 3DP-BSG for BCS in breast cancer patients receiving NST, the original tumor area can be identified and marked directly on the breast, which is useful for surgery. Trial Registration: Clinical Research Information Service (CRIS) Identifier Number: KCT0002272. First registration number and date: No. 1 (27/04/2016).