Colorectal carcinoma is a common malignancy. Alternative treatment strategies, including nano-based formulations, show remarkable promise to improve the efficacy of chemotherapy. The soy isoflavone daidzein has attracted attention for its anti-cancer activity. The aim of this study was to investigate the effects of daidzein nanosuspension (DZ-NS) with 5-fluorouracil (5-FU) on apoptosis and inflammation induced in human colorectal adenocarcinoma cells (Caco-2). Cytotoxicity, total antioxidant/oxidant status (TAS, TOS), and the expression of proteins associated with apoptosis and inflammation were analyzed by the MTT, ELISA and qRT-PCR, respectively. DZ-NS showed a strong cytotoxicity (with lower IC50) compared to daidzein. The combination of 5-FU and DZ-NS treatment showed a synergistic antiproliferative effect without increasing oxidative stress. The combination showed a significant increase in p53 levels and a significant decrease in Bcl-2, IL-6, TNF-α and MMP-9 levels. These results were confirmed at the molecular level. In conclusion, DZ-NS with 5-FU treatment reduced the proliferation and inflammation and increased apoptosis of Caco-2 cells without oxidative stress. The current study shows that this combination also elucidates several signaling pathways, including effects on oxidative stress, apoptosis, and inflammation. The integration of this nanotechnology approach into traditional chemotherapy will form the basis of an innovative strategy.