Abstracttrans-Fatty acids (TFAs) are unsaturated fatty acids containing at least one carbon–carbon double bond in trans configuration, which are classified into two groups according to their food source: industrial TFAs (iTFAs) and ruminant TFAs (rTFAs). Previous epidemiological evidence has demonstrated a preferential association of iTFAs, rather than rTFAs, with various diseases including cardiovascular diseases. However, it is still unknown how iTFAs exert their specific toxicity and what effective treatments are available to mitigate their toxicity. Here, we performed a comprehensive toxicological assessment of TFAs based on the toxicity mechanism that we established previously. We found that iTFAs including elaidic acid (EA), but not other types of fatty acids including rTFAs, had a strong pro-apoptotic effect upon treatment of extracellular ATP, a damage-associated molecular pattern that induces apoptosis through the apoptosis signal-regulating kinase 1 (ASK1)-p38 MAP kinase pathway. We also found that polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), potently suppressed EA-dependent increase in ASK1 activation and apoptosis. These results demonstrate that iTFAs specifically exert toxicity by targeting ASK1, and that PUFAs serve as their effective suppressor. Our study provides a molecular basis for risk assessment of foods, and for new prevention and treatment strategies for TFA-related diseases.