Background The evaluation of prognostic factors in rectal carcinoma patients has important clinical significance. P53 status and the Ki-67 index have served as prognostic factors in rectal carcinoma. Amide proton transfer (APT) imaging has shown great potential in tumor diagnosis. However, few studies reported the value of APT imaging in evaluating p53 and Ki-67 status of rectal carcinoma. Purpose To investigate the feasibility of amide proton transfer MRI in assessing p53 and Ki-67 expression of rectal adenocarcinoma, and compare it with conventional diffusion-weighted imaging (DWI). Study Type Retrospective. Population Forty-three patients with rectal adenocarcinoma (age: 34-85 years). Field Strength/Sequence 3T/APT imaging using a 3D turbo spin echo (TSE)-Dixon pulse sequence with chemical shift-selective fat suppression, 2D DWI, and 2D T-2-weighted TSE. Assessment Mean tumor APT signal intensity (SImean) and apparent diffusion coefficient (ADC(mean)) were measured. Traditional tumor pathological analysis included WHO grades, pT (pathologic tumor) stages, and pN (pathologic node) stages. Expression levels of p53 and Ki-67 were determined by immunohistochemical assay. Statistical Tests One-way analysis of variance (ANOVA); Student'st-test; Spearman's correlation coefficient; receiver operating characteristic (ROC) curve analysis. Results High-grade tumors, more advanced stage tumors, and tumors with lymph node involvement had higher APT SI(mean)values: high grade (n =15) vs. low-grade (n =28),P < 0.001; pT2 (n =10) vs. pT3 (n =20) vs. pT4 (N =13),P= 0.021; pN0 (n =24) vs. pN1-2 (n =19),P= 0.019. ADC(mean)differences were found in tumors with different pT stage: pT2 (n =10) vs. pT3 (n =20) vs. pT4 (N =13),P= 0.013, but not in tumors with different histologic grade: high grade (n =15) vs. low-grade (n =28),P= 0.3536; or pN stage: pN0 (n =24) vs. pN1-2 (n =19),P= 0.624. Tumor with p53 positive status had higher APT SI(mean)than tumor with negative p53 status (2.363 +/- 0.457 vs. 2.0150 +/- 0.3552,P= 0.014). There was no difference in ADC(mean)with p53 status (1.058 +/- 0.1163 10(-3) mm(2)/s vs. 1.055 +/- 0.128 10(-3) mm(2)/s,P= 0.935). APT SI(mean)and ADC(mean)were significantly different in tumors with low and high Ki-67 status (1.7882 +/- 0.11386 vs. 2.3975 +/- 0.41586,P < 0.001; 1.1741 +/- 0.093 10(-3) mm(2)/s vs. 1.0157 +/- 0.10459 10(-3) mm(2)/s,P < 0.001, respectively). APT SI(mean)exhibited a positive correlation with p53 labeling index and Ki-67 labeling index (r =0.3741,P= 0.0135;r =0.7048;P < 0.001, respectively). ADC(mean)showed no correlation with p53 labeling index, but a negative correlation with Ki-67 labeling index (r = -0.5543,P < 0.0001). ROC curves demonstrated that APT SI(mean)had significantly higher diagnostic ability for differentiation of high Ki-67 expression of rectal adenocarcinoma than ADC(mean)(81.2% vs. 78.12%, 90.91% vs. 63.64;P < 0.001 vs.P= 0.017), while no difference was found in predicting p53 status (92.86% vs. 71.4%, 53.33% vs. 66.7%;P < 0.001 vs.P= 0.0471). Data Conclusion APT SI(mean)was related to p53 and Ki-67 expression levels in rectal adenocarcinoma. APT imaging may serve as a noninvasive biomarker for assessing genetic prognostic factors of rectal adenocarcinoma. Level of Evidence 3 Technical Efficacy Stage 2