In the present study, we investigated NF-κB p65 phosphorylated at Serine-536 (phospho-Ser536-p65) in rectal cancer and its relationship to radiotherapy (RT) and clinicopathological and biological factors. Expression of phospho-Ser536-p65 was examined by using immunohistochemistry in 141 primary rectal cancers, 149 normal mucosa specimens and 48 metastases in the lymph nodes, from rectal cancer patients randomized to received RT or not. The expression of phospho-Ser536-p65 in the cytoplasm increased from normal mucosa to primary tumor (p<0.0001, for both RT and non-RT groups). The expression did not further increased from primary tumor to metastases in the either group (p>0.05). We found that the expression of phospho-Ser536-p65 was positively related to or tended to be positively related to expression of TEM1 (p=0.02), FXYD-3 (p=0.0006), PRL (p=0.02), p73 (p=0.048) and MAC30 (p=0.051) in the RT group but there were no such relationships in the non-RT group (p>0.05). The expression of the phospho-Ser536-p65 was not related to clinicopathological factors including survival (p>0.05). The increased expression of phospho-Ser536-p65 may be involved in rectal cancer development. After RT, the expression of NF-κB seems to be positively related to the biological factors which associated with more malignant features of tumors. However, we did not find that the phospho-Ser536-p65 was directly related to clinical response of RT.