Objective . This study aims to investigate the potential of mustard oil-induced reduction in acetylcholine expression as a means to delay the progression of colon cancer within the internal environment. Methods . The study design in this research involved both in vitro cellular experiments and in vivo animal experiments to employ mustard oil to modulate acetylcholine expression levels and evaluate its impact on colon cancer. Cellular experiments involved the introduction of six concentrations of acetylcholine (10(-2), 10(-3), 10(-4), 10(-5), 10(-6), and 10(-7) mol/L) into colon cancer cell cultures to monitor cell proliferation. Animal experiments encompassed the subcutaneous CT26 colon cancer cells implantation into 28 Balb/c mice, divided into experimental and control groups. After tumor establishment, both groups were fed standard diets for two weeks. Serum acetylcholine concentrations were measured from eye blood samples. Additionally, Balb/c mice were inoculated with CT26-derived colon cancer cells and further categorized into experimental and control groups. A total of 14 mice comprised each group, with experimental mice fed mustard oil and control mice fed soybean oil. Post two weeks, serum acetylcholine expression in both groups was assessed. After sacrifice, subcutaneous tumors were excised, and tumor dimensions were measured using a Vernier scale. Results . Acetylcholine concentration augmentation in the culture medium corresponded to gradual cell proliferation escalation, peaking at 10(-5) mol/L, exhibiting statistical significance. Comparative analysis revealed significantly elevated relative acetylcholine expression levels in Balb/c mice with tumor-bearing colon cancers compared to normal Balb/c mice. Experimental group mice exhibited substantially lower serum acetylcholine concentrations than control group mice. Mustard oil administration effectively curtailed acetylcholine expression in normal Balb/c mice, consequently retarding tumor growth. These findings underscore mustard oil's potential to diminish serum acetylcholine expression, thereby delaying colon cancer progression. Conclusions . This study suggests that mustard oil's modulation of acetylcholine expression within the internal environment holds the potential for impeding colon cancer growth.