Accurate measurement of glomerular filtration rate (GFR) is essential for optimal decision making in many clinical settings of renal failure. We aimed to show that GFR can be accurately measured using compartmental tracer kinetic analysis of 18F-fluoride dynamic PET/CT. Twenty-three male Sprague-Dawley rats of three experimental groups (cyclosporine-administered [n = 8], unilaterally nephrectomized [n = 8], and control [n = 7]) underwent simultaneous 18F-fluoride dynamic PET/CT and reference 51Cr-EDTA GFR (GFRCrEDTA) test at day 0 and post-intervention day 3. 18F-fluoride PET GFR (GFRF-PET) was calculated by multiplying the influx rate and functional kidney volume in a single-tissue-compartmental kinetic model. Within-test repeatability and between-test agreement were evaluated by intraclass correlation coefficient (ICC) and Bland-Altman analysis. In the control group, repeatability of GFRF-PET was excellent (ICC = 0.9901, repeatability coefficient = 12.5%). GFRF-PET significantly decreased in the renally impaired rats in accordance with respective GFRCrEDTA changes. In the pooled population, GFRF-PET agreed well with GFRCrEDTA with minimal bias (-2.4%) and narrow 95% limits of agreement (-25.0% to 20.1%). These data suggest that the single-compartmental kinetic analysis of 18F-fluoride dynamic PET/CT is an accurate method for GFR measurement. Further studies in humans are warranted.