The iturin produced by Bacillus subtilis was previously found to exhibit significant antifungal activities, showing potential for application to food to protect it from spoilage and decay. However, its safe level must be evaluated before it can be used in foods consumed by mammals. Therefore, we carried out acute toxicity (7 days) and subacute toxicity (28 days) evaluations by intragastric administration of the iturin at 5000 mg per kg (bw) and 2000 mg per kg (bw) to Kunming mice. At the end of the subacute toxicity evaluation, the blood parameters of the mice were evaluated, and the visceral organs and intestinal flora were examined. No acute toxicity was found for 5000 mg per kg (bw) iturin and no significant negative effects were found for 2000 mg per kg (bw) iturin on blood parameters or organ functions. A more important observation was that the application of the iturin at the concentration of 2000 mg per kg (bw) showed great enhancement of the abundance of probiotics in the intestinal microflora. The iturin also showed inhibitory effects on cancer cells HepG2, Caco-2, MCF-7, A549, and BIU-87, at the level of 30 μM. The results indicate that the B. subtilis iturin has the potential to be developed as a functional food additive with antitumor potential and the capability to enhance the abundance of probiotics in the intestinal microflora. This study indicates a new possible source of functional food additives.