Colon adenocarcinoma is one of the most common fatal malignancies in Western countries. Progression of this cancer is dependent on tumor microenvironmental signaling molecules such as transforming growth factor-beta (TGF-beta) or acetylcholine (ACh). The present study was conducted to assess the influence of recombinant human transforming growth factor (rhTGF)-beta1 or ACh on nitric oxide (NO) and interleukin-1beta (IL-1beta) secretion by three human colon adenocarcinoma cell lines: HT29, LS180, and SW948, derived from different grade tumors (Duke's stage). The cells were cultured in 2D and 3D (spheroids) conditions. Colon carcinoma cells exhibited different sensitivities to rhTGF-beta1 or ACh dependent on the tumor grade and the culture model. ACh exhibited significant inhibitory effects towards NO, endothelial nitric oxide synthase (eNOS), and IL-1beta secretion especially by tumor cells derived form Duke's C stage of colon carcinoma. rhTGF-beta1 also decreased NO, IL-1beta, and eNOS expression, but its effect was lower than that observed after the administration of ACh. The inhibition of NO and IL-1beta production was more striking in 3D tumor spheroids than in 2D culture monolayers. Taken together, the TGF-beta1-ACh axis may regulate colon carcinoma progression and metastasis by altering NO secretion and influence inflammatory responses by modulating IL-1beta production.