Abstract Background and Aims Renal fibrosis (RF) being the most important pathological change in the progression of CKD, is currently assessed by the evaluation of a biopsy. This present study aimed to apply a novel functional MRI (fMRI) protocol named amide proton transfer weighting (APTw) to evaluate RF non-invasively. Method Male Sprague-Dawley (SD) rats were initially subjected to bilateral kidney ischemia/reperfusion injury (IRI), unilateral ureteral obstruction (UUO) and Sham operation, respectively. All rats underwent APT mapping on the 7th and the 14th day after operation. Besides, 26 patients undergoing renal biopsy at the Nephrology Department of Shanghai Tongji Hospital between July 2022 and May 2023. Patients underwent APT and apparent diffusion coefficient (ADC) mappings within 1 week before biopsy. MRI results of both patients and rats were calculated by comparing with gold standard histology for fibrosis assessment. Results In animal models, the cortical APT (cAPT) and medullary APT (mAPT) values were positively correlated with the degree of renal fibrosis. Compared to the sham group, IRI group showed significantly increased cAPT and mAPT values on the 7th and 14th day after surgery, but no group differences were found in ADC values. Similar results were found in human patients. Cortical/medullary APT values were significantly increased in patients with moderate-to-severe fibrosis than patients with mild fibrosis. ROC curve analysis indicated that APT value displayed a better diagnostic value for RF. Furthermore, combination of cADC and cAPT improved fibrosis detection by imaging variables alone (p < 0.1). Conclusion APT values had better diagnostic capability at early stage of RF compared to ADC values, and the addition of APT imaging to conventional ADC will significantly improve the diagnostic performance for predicting kidney fibrosis.