Prostatic zinc content is a known biomarker for discriminating normal healthy tissue from benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Given that zinc content is not readily measured without a tissue biopsy, we have been exploring noninvasive imaging methods to detect these diagnostic differences using a zinc-responsive MRI contrast agent. During imaging studies in mice, we observed that a bolus of glucose stimulates secretion of zinc from the prostate of fasted mice. This discovery allowed the use of a Gd-based zinc sensor to detect differential zinc secretion in regions of healthy versus malignant prostate tissue in a transgenic adenocarcinoma mouse model of PCa. Here, we used a zinc-responsive MRI agent to detect zinc release across the prostate during development of malignancy and confirm the loss of total tissue zinc by synchrotron radiation X-ray fluorescence (μSR-XRF). Quantitative μSR-XRF results show that the lateral lobe of the mouse prostate uniquely accumulates high concentrations of zinc, 1.06 ± 0.08 mM, and that the known loss of zinc content in the prostate is only observed in the lateral lobe during development of PCa. Additionally, we confirm that lesions identified by a loss of zinc secretion indeed represent malignant neoplasia and that the relative zinc concentration in the lesion is reduced to 0.370 ± 0.001 mM. The μSR-XRF data also provided insights into the mechanism of zinc secretion by showing that glucose promotes movement of zinc pools (∼1 mM) from the glandular lumen of the lateral lobe of the mouse prostate into the stromal/smooth muscle surrounding the glands. Co-localization of zinc and gadolinium in the stromal/smooth muscle areas as detected by μSR-XRF confirm that glucose initiates secretion of zinc from intracellular compartments into the extracellular spaces of the gland where it binds to the Gd-based agent and albumin promoting MR image enhancement.