Colitis associated cancer (CAC) is the colorectal cancer (CRC) subtype that is associated with bowel disease such as ulcerative colitis (UC). The data on role of NF- κB signaling in development and progression of CAC were derived from preclinical studies, whereas data from human are rare. The aim of this work was to study the contribution of NF- κB pathway during UC and CAC, as well as the immunomodulatory effect of all-trans retinoic acid (AtRA). We analyzed the expression of NOS2, TNF- α, TLR4, and NF- κB, in colonic mucosa. We also studied NO/TNF- α modulation by LPS in colonic mucosa pretreated with AtRA. A marked increase in TLR4, NF- κB, TNF- α, and NOS2 expression was reported in colonic mucosa. The relationship between LPS/TLR4 and TNF- α/NO production, as well as the role of NF- κB signaling, was confirmed by ex vivo experiments and the role of LPS/TLR4 in NOS2/TNF- α induction through NF- κB pathway was suggested. AtRA downregulates NOS2 and TNF- α expression. Collectively, our study indicates that AtRA modulates in situ LPS/TLR4/NF- κB signaling pathway targeting NOS2 and TNF- α expression. Therefore, we suggest that AtRA has a potential value in new strategies to improve the current therapy, as well as in the clinical prevention of CAC development and progression.