Background: To obtain a clear resection margin during breast-conserving surgery (BCS) after neoadjuvant chemotherapy (NCT), accurate targeting of the location and assessment extent of the tumor are essential. However, conventional targeting methods such as USG/MMG guided hook wire insertion have disadvantages in that they could only localize the tumor, not reflecting its extent. To assess this problem, we developed an MRI-based 3D printed breast surgical guide (3DP-BSG) and conducted a multicenter randomized clinical study to prove its clinical effectiveness. Methods: In this multicenter, randomized (1:1), controlled trial (KCT0004469), we assigned 566 patients who underwent NCT and planned to undergo BCS, to use customized 3DP-BSG for lumpectomy, or not to use 3DP-BSG for lumpectomy. We assumed that 3DP-BSG group achieve non-inferior outcomes compared with control group with conventional targeting method. The primary endpoint was the margin positivity of the first resected margin. Under the assumption that the proportion of margin positivity would be 23% in both groups, a total of 438 patients were calculated to have 80% power to establish non-inferiority with a margin of 10% at a one-sided significance level of 0.05. Efficacy was assessed in the intention-to-treat (ITT) population. Results: Between 2019 and 2021, 282 (49.8%) patients were assigned to 3DP-BSG group, and 284 (50.2%) patients were assigned to control group with conventional targeting method. The median age was 51 years. In the ITT analysis, first resected margin was positivity in 5 (2.1%) of 235 patients in 3DP-BSG group and 7 (2.9%) of 244 patients in control group (difference -1.15 [95% CI -3.58 to -1.28], pnon-inferiority <.0001). In the analysis of population excluding patients assessed as complete response (CR), first resected margin was positivity in 5 (3.40%) of 147 patients in 3DP-BSG group and 7 (4.8%) of 145 patients in control group (difference -1.43 [95% CI -5.25 to -2.40], pnon-inferiority <.0001). Most of the cases, marked areas with 3DP-BSG were partially different from those with conventional targeting method, and there were some cases where completely different areas were marked. In most of these cases, pathological CR made it difficult to compare the accuracy of the targeting methods, but in one case, a malignant tumor was diagnosed only in the 3DP-BSG targeting region. Even lesions that have been incorrectly targeted by conventional methods can be properly removed using 3DP-BSG, which is believed to be helpful in surgery. Conclusions: Lumpectomy using 3DP-BSG showed non-inferiority compared to the conventional targeting group, and the surgeon's satisfaction was higher by quantitatively presenting the extent of initial tumor. Clinical trial information: KCT0004469.