Objectives: Renal mitochondrial damage are recognized as a major pathogenic events in chronic kidney disease (CKD). However, optimal imaging methods to assess renal mitochondrial dysfunction are not established. Our aim was to investigate the potential value of renal imaging with 99mTc-sestamibi (MIBI), which localizes primarily in mitochondria depending on membrane potential. Methods: Six newly diagnosed CKD patients and 21 healthy volunteers(HV) were prospectively enrolled. All underwent 99mTc-DTPA dynamic renal scan followed by MIBI dynamic renal scan and SPECT/CT. On dynamic scintigraphy, radioactivity concentration of the kidneys was measured as % injected doses (%ID) at 2-3 min with time-activity curves. Kidneys were manually delineated on SPECT/CT and quantitative analyses were performed. We used logistic regression to predict CKD and the area under the curve (AUC) of the receiver operator characteristic curve was calculated. Results: Patterns of elimination were different between MIBI and DTPA renograms as MIBI washout was slower than DTPA (T1/2, 16.6 min vs. 9.9 min, p<0.0001) with plateau of MIBI reached around 15 min. In comparison between CKD and HV groups, CKD group showed lower estimated glomerular filtration rate (eGFR, 87.1 vs. 107.2 ml/min, p = 0.02) and lower renal concentration of MIBI than HV group, e.g. %ID (6.2% vs. 8.7%, p = 0.01), SUVmax (11.8 vs. 15.3, p = 0.05), and SUVmean (6.1 vs. 8.6, p = 0.01). However, DTPA parameters were not significantly different between groups.  In multivariable analysis, SUVmean was the significant parameter for CKD, as decreasing SUVmean was associated with an increased likelihood of CKD (p = 0.03, Odds ratio = 3.94 (95.0% CI 1.18-13.16)). The eGFR model and the MIBI model yielded an AUC of 0.73 and 0.88, respectively. Combining eGFR and MIBI model showed best performance (AUC 0.95, p <0.001).  Conclusions: Renal 99mTc-MIBI uptake could be a new imaging biomarker for diagnosing renal mitochondrial dysfunction.