In this paper, we report cases of two patients with vaginal tumor who underwent interstitial brachytherapy (ISBT), using three-dimensional (3D)-printed personalized templates designed inversely from computed tomography (CT) or magnetic resonance (MR) images. Patient 1 presenting with vaginal vault recurrence was planned to receive whole pelvis external beam radiotherapy (EBRT) followed by ISBT. The tumor invaded the paracolpium; thus, we planned to administer ISBT to include the tumor and vaginal membrane. A template was designed with holes for plastic needle applicator insertion considering the appropriate direction based on pre-treatment medical images. Patient 2 presenting with vaginal cancer was scheduled to receive EBRT and ISBT because of a paracolpium invasion. Before ISBT, MR imaging was performed with vaginal cylinder inserted in the patient's vagina. By measuring the length of the tumor manually and projecting the tumor orthogonally to a plane parallel to the bottom surface of the cylinder applicator, a template was designed. Computer-aided design software was used for planning both templates. Polycarbonate/acrylonitrile-butadiene-styrene resin was selected as material of the templates. Patient 1 received 4-fraction ISBT one week apart. A mean of 10 applicators were inserted through the holes of the template in an average of 9 minutes (range, 5-15 minutes). All applicators were inserted toward the planned directions. Median minimum dose covering 90% (D) of the clinical target volume (CTV) was 634 cGy. Patient 2 underwent three-fraction irradiation twice daily at 6-hour interval. All applicators were inserted through the inside of the template. The median D of the CTV was 703 cGy. No grade 3 or higher toxicity were found in both series. 3D-printed templates designed using medical images are useful, especially for ISBT of vaginal tumors. Further verification of clinical indications, design of templates, and manufacturing process are needed.