Accurate preoperative diagnosis for gastrointestinal stromal tumor (GIST) was obtained using the endoscopic approach in about 50% of the cases. Preoperative assessment is quite important in reproductive women for the treatment of pelvic tumor. We report the first case of GIST of the rectum that was accurately diagnosed with preoperative needle biopsy from the vaginal wall. The case was a 38-year-old woman who presented with a history of genital bleeding. Internal examination revealed a round and elastic mass (7 x 7 cm) in the left pelvic cavity. The biopsy specimen from the left sidewall of the vagina using a manually manipulated biopsy needle showed strong reaction for CD34 and CD117 (c-KIT). The patient underwent low anterior resection of the rectum, tumor resection, and partial resection of the posterior vagina. Preoperative biopsy with vaginal approach might be helpful to make a diagnosis for pelvic tumor of unknown origin and unknown malignant potential.