To evaluate whether aggressive characteristics of rectal cancer can be predicted by the apparent diffusion coefficient (ADC) obtained using readout-segmented echo-planar imaging (rs-EPI) diffusion-weighted magnetic resonance. We enrolled one hundred and fifteen patients. The image quality of ADC maps by rs-EPI was compared with that by traditional single-shot echo-planar imaging (ss-EPI), and ADC measurement was performed on the rs-EPI based ADC maps. Differences in ADC values of tumors grouped according to differentiation grade, clinical T stage and plasmatic carcinoembryonic antigen (CEA) level were tested. The correlation between each aggressive characteristic and the corresponding ADC values was evaluated. The image quality of ADC maps obtained by rs-EPI was superior toss-EPI (P < 0.05). The ADC values of tumor were categorized based on the following differentiation grades: poor (0.89 ± 0.12 × 10-3 mm2/s), moderate (1.13 ± 0.25 × 10-3 mm2/s), and good (1.31 ± 0.19 × 10-3 mm2/s); P < 0.001. Tumors with lower differentiation grades corresponded to lower ADC values (r = 0.59, P < 0.001). However, ADC differences were not observed in different clinical T stage (P = 0.22) and plasmatic CEA level (P = 0.38). Rs-EPI sequence-based ADC values represent a potential imaging marker for the aggressive rectal cancer characteristics.