This study was conducted to evaluate the toxic effects of an azo dye carmoisine widely used in foods and to investigate its relation to carcinogenicity. Carmoisine administered into mice orally in four different doses as control, low, medium, and high equivalent to 0, 4, 200, and 400 mg/kg bw, respectively, for 120 days. The key toxicological endpoint was observed including animal body weight, organ weights, hematology, biochemistry, and molecular biology assessment. The body weights of medium‐ and high‐dose carmoisine‐treated mice group were significantly decreased as compared to the control mice group. Platelet, white blood cell and monocyte counts of treated group were considerably higher, while Hb and red blood cell counts were drastically lower than the control group. The biochemical parameters such as serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, globulin, urea, and creatinine level were significantly increased, while serum cholesterol level was decreased after treatment as compared to the control. RT‐PCR results showed that expression of Bcl‐x and PARP gene was intensively increased, whereas expression of p⁵³ gene was decreased in the mouse liver tissues treated with carmoisine. This study revealed that high‐dose (400 mg/kg bw) treatment of carmoisine was attributable to renal failure and hepatotoxicity. It also would be suspected as a culprit for liver oncogenesis.