Functional imaging modalities like Diffusion-weighted imaging are increasingly used to predict tumor behavior like cellularity and vascularity in different tumors. Histogram analysis is an emergent imaging analysis, in which every voxel is used to obtain a histogram and therefore statistically information about tumors can be provided. The purpose of this study was to elucidate possible associations between ADC histogram parameters and several immunhistochemical features in rectal cancer. Overall, 11 patients with histologically proven rectal cancer were included into the study. There were 2 (18.18%) females and 9 males with a mean age of 67.1 years. KI 67-index, expression of p53, EGFR, VEGF, and Hif1-alpha were semiautomatically estimated. The tumors were divided into PD1-positive and PD1-negative lesions. ADC histogram analysis was performed as a whole lesion measurement using an in-house matlab application.Spearman's correlation analysis revealed a strong correlation between EGFR expression and ADCmax (p=0.72, P=0.02). None of the vascular parameters (VEGF, Hif1-alpha) correlated with ADC parameters. Kurtosis and skewness correlated inversely with p53 expression (p=-0.64, P=0.03 and p=-0.81, P=0.002, respectively). ADCmedian and ADCmode correlated with Ki67 (p=-0.62, P=0.04 and p=-0.65, P=0.03, respectively). PD1-positive tumors showed statistically significant lower ADCmax values in comparison to PD1-negative tumors, 1.93 ± 0.36 vs 2.32 ± 0.47×10−3mm2/s, p=0.04.Several associations were identified between histogram parameter derived from ADC maps and EGFR, KI 67 and p53 expression in rectal cancer. Furthermore, ADCmax was different between PD1 positive and PD1 negative tumors indicating an important role of ADC parameters for possible future treatment prediction.