The purpose of this study was to develop an aggressive locally advanced orthotopic prostate cancer model for assessing high-dose image-guided radiation therapy combined with biological agents. For this study, we used a modified human prostate cancer (PCa) cell line, PC3, in which we knocked down a tumor suppressor protein, DAB2IP (PC3‑KD). These prostate cancer cells were implanted into the prostate of nude or Copenhagen rats using either open surgical implantation or a minimally invasive procedure under ultrasound guidance. We report that: i) these DAB2IP-deficient PCa cells form a single focus of locally advanced aggressive tumors in both nude and Copenhagen rats; ii) the resulting tumors are highly aggressive and are poorly controlled after treatment with radiation alone; iii) ultrasound-guided tumor cell implantation can be used successfully for tumor development in the rat prostate; iv) precise measurement of the tumor volume and the treatment planning for radiation therapy can be obtained from ultrasound and MRI, respectively; and v) the use of a fiducial marker for enhanced radiotherapy localization in the rat orthotopic tumor. This model recapitulates radiation-resistant prostate cancers which can be used to demonstrate and quantify therapeutic response to combined modality treatments.