AbstractAmyotrophic lateral sclerosis (ALS) is a fatal, adult‐onset, neurodegenerative disease. The transactivating response region DNA binding protein 43 (TDP‐43) p.Q331K mutation (TDP‐43 Q331K) has previously been identified in ALS as a disease‐causing mutation with neurotoxicity. SecinH3, a cytohesin inhibitor, has neuroprotective effects against mutant superoxide dismutase 1 (SOD1) toxicity. However, whether SecinH3 protects against mutant TDP‐43 p.Q331K protein toxicity and its potential molecular mechanisms have not yet been investigated. To determine whether TDP‐43 Q331K induces neuronal toxicity, SH‐SY5Y, a human derived neuronal cell line were selected as an in vitro model of neuronal function. SH‐SY5Y cells were transiently transfected with TDP‐43 wild‐type or TDP‐43 Q331K. Remarkably, TDP‐43 Q331K induced neuronal damage via endoplasmic reticulum (ER) stress‐mediated apoptosis and the impairment of the autophagic flux. SecinH3 was demonstrated to successfully attenuate the TDP‐43 Q331K‐induced neuronal toxicity by suppressing ER stress‐mediated apoptosis and enhancing the autophagic flux. Taken together, our in vitro study provided evidence that SecinH3 exerts neuroprotective effects against TDP‐43 Q331K‐mediated neuronal toxicity and was able to elucidate its mode of action. SecinH3 could, therefore, be considered a promising candidate as a therapeutic agent of ALS. © 2018 IUBMB Life, 71(1):192–199, 2019