Colon cancer (CC) is among the most common malignant diseases with a dismal survival. Tumor necrosis factor-alpha (TNF-α) has been identified as a therapeutic target in various cancers, and anti-TNF-α treatment has shown promising effects in different cancer models. However, if TNF-α can be targeted in CC, the therapeutic values of anti-TNF-α treatment in CC remain unknown. Our study indicated that TNF-α is highly expressed in CC cell lines and patient tumor samples. High expression of TNF-α is an independent adverse prognosticator of CC. Targeting the TNF-α by its antibody infliximab induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and enhanced apoptosis leading to cell death. The combination of infliximab with 5-fluorouracil showed better responses in vitro and in vivo than 5-fluorouracil alone. In conclusion, this study identified TNF-α as a target of CC and anti-TNF-α treatment synergized with chemotherapy leading to a better outcome in preclinical models.