PurposeTo assess the performance of two point (2-pt) Dixon-based chemical exchange saturation transfer (CEST) imaging for fat suppression in renal transplant patients. MethodsThe 2-pt Dixon-based CEST MRI was validated in an egg-phantom and in fourteen renal transplant recipients (5 females and 9 males; age range: 23–78 years; mean age: 51 ± 16.8). All CEST experiments were performed on a 3 T clinical MRI scanner using a dual-echo CEST sequence. The 2-pt Dixon technique was applied to generate water-only CEST images at different frequency offsets, which were further used to calculate the z-spectra. The magnetization transfer ratio asymmetry (MTRasym) values in the frequency ranges of hydroxyl, amine and amide protons were estimated in the renal cortex and medulla. ResultsResults of the in vitro experiments suggest that the 2-pt Dixon technique enables effective fat peak removal and does not introduce additional asymmetries to the z-spectrum. Accordingly, our results in vivo show that the fat-corrected amide proton transfer (APT) effect in the kidney is significantly higher compared to that obtained from the CEST data acquired close to the in-phase condition both in the renal cortex (−0.1 [0.7] vs. −0.7 [1.2], P = 0.029) and medulla (0.3 [0.8] vs. 0.01 [1.3], P = 0.049), indicating that the 2-pt Dixon-based CEST method increases the specificity of the APT contrast by correcting the fat-induced artifacts. ConclusionCombination of the dual-echo CEST acquisition with Dixon post-processing provides effective water-fat separation, allowing more accurate quantification of the APT CEST effect in the transplanted kidney.