Diallyl trisulfide (DATS) is one of the main sulfide-containing compounds in garlic oil and has been shown to have a variety of biological activities such as anti-tumor, inhibition of inflammation, and immune regulation. However, there is very little information available regarding its toxicological safety. To elucidate the toxicity mechanism and find the early warning biomarker of DATS, this study conducted acute and subacute toxicity experiments according to the OECD guidelines. The potential serum biomarker and toxicity mechanism of DATS were explored by proteomics.