Purpose: To compare four region-of-interest (ROI) protocols for apparent diffusion coefficient (ADC) quantifications derived from reduced field-of-view diffusion-weighted imaging (rDWI) in histological characterization of rectal cancer. Materials and methods: Forty-nine patients with rectal cancer underwent rDWI at 3.0 T. Two readers independently performed mean and minimum ADC measurements using four different ROI positioning protocols (whole tumor volume [WTV], single-slice [SS], three-slices observer-based sampling [TSOB] and three-slices predefined sampling [TSPD]). Inter-observer variation was evaluated. Mean and minimum ADC values obtained from each method were compared in terms of different histological factors of rectal cancer, and their diagnostic abilities were assessed by receiver operating characteristic curve (ROC) analysis. The corresponding times for ADC measurements were recorded and compared between ROI methods. Results: The inter-observer agreement was excellent for ADC values obtained by two readers using the four ROI methods (ICC range, 0.906-0.994). Mean and minimum ADC values by WTV method were significantly higher and lower than other methods respectively for both readers (P < 0.05). The AUCs of mean ADC measurements for assessment of well-differentiated tumors, T2 stage tumors and NO status (no lymph node metastasis) (0.936, 0.840 and 0.714) were greater by WTV method than those by SS method (0.782, 0.761 and 0.677). The WTV method required longer measurement time than other ROI methods (P < 0.001). Conclusion: ADC measurements based on rDWI were influenced by ROI positioning protocols. rDWI technique had diagnostic value for histological characterization of rectal cancer, using WTV method with overall best inter-observer reproducibility, but with the longest measurement time.