| Summary: | (1) Background: Colon cancer is one of the most common cancer types, and treatment options, unfortunately, do not continually improve the survival rate of patients. With the unprecedented development of nanotechnologies, nanomedicine has become a significant direction in cancer research. Indeed, chemotherapeutics with nanoparticles (NPs) in cancer treatment is an outstanding new treatment principle. (2) Methods: Fe<sub>3</sub>O<sub>4</sub> NPs were synthesized and characterized. Caco-2 colon cancer cells were treated during two different periods (24 and 72 h) with Fe<sub>3</sub>O<sub>4</sub> NPs (6 μg/mL), various concentrations of 5-FU (4–16 μg/mL), and Fe<sub>3</sub>O<sub>4</sub> NPs in combination with 5-FU (4–16 μg/mL) (Fe<sub>3</sub>O<sub>4</sub> NPs + 5-FU). (3) Results: The MTT assay showed that treating the cells with Fe<sub>3</sub>O<sub>4</sub> NPs + 5-FU at 16 µg/mL for 24 or 72 h decreased cell viability and increased their LDH release (<i>p</i> < 0.05 and <i>p</i> < 0.01, respectively). Furthermore, at the same treatment concentrations, total antioxidant capacity (TAC) was decreased (<i>p</i> < 0.05 and <i>p</i> < 0.01, respectively), and total oxidant status (TOS) increased (<i>p</i> < 0.05 and <i>p</i> < 0.01, respectively). Moreover, after treatment with Fe<sub>3</sub>O<sub>4</sub>-NPs + 5-FU, the IL-10 gene was downregulated and PTEN gene expression was upregulated (<i>p</i> < 0.05 and <i>p</i> < 0.01, respectively) compared with those of the control. (4) Conclusions: Fe<sub>3</sub>O<sub>4</sub> NPs exert a synergistic cytotoxic effect with 5-FU on Caco-2 cells at concentrations below the active drug threshold levels.
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