TQFL12 is a novel derivative designed and synthesized on the basis of Thymoquinone (TQ) which is extracted fromNigella sativaseeds. We have demonstrated that TQFL12 was more effective in the treatment of TNBC than TQ. In order to directly reflect the acute toxicity of TQFL12 in vivo, in this study, we designed, synthesized, and compared it with TQ. The mice were administered drugs with different concentration gradients intraperitoneally, and death was observed within one week. The 24 h median lethal dose (LD50) of TQ was calculated to be 33.758 mg/kg, while that of TQFL12 on the 7th day was 81.405 mg/kg, and the toxicity was significantly lower than that of TQ. The liver and kidney tissues of the dead mice were observed by H&E staining. The kidneys of the TQ group had more severe renal damage, while the degree of the changes in the TQFL12 group was obviously less than that in the TQ group. Western blotting results showed that the expressions of phosphorylated levels of adenylate-activated protein kinase AMPKα were significantly up-regulated in the kidneys of the TQFL12 group. Therefore, it can be concluded that the acute toxicity of TQFL12 in vivo is significantly lower than that of TQ, and its anti-toxicity mechanism may be carried out through the AMPK signaling pathway, which has a good prospect for drug development.