The T-2 toxin and deoxynivalenol (DON), as the most concerned members of trichothecenes, induce cellular stress responses and various toxic effects. Stress granules (SGs) are rapidly formed in response to stress and play an important role in the cellular stress response. However, it is not known whether T-2 toxin and DON induce SG formation. In this study, we found that T-2 toxin induces SG formation, while DON surprisingly suppresses SG formation. Meanwhile, we discovered that SIRT1 co-localized with SGs and regulated SG formation by controlling the acetylation level of the SG nucleator G3BP1. Upon T-2 toxin, the acetylation level of G3BP1 increased, but the opposite change was observed upon DON. Importantly, T-2 toxin and DON affect the activity of SIRT1 via changing NAD+ level in a different manner, though the mechanism remains to be clarified. These findings suggest that the distinct effects of T-2 toxin and DON on SG formation are caused by changes in the activity of SIRT1. Furthermore, we found that SGs increase the cell toxicity of T-2 toxin and DON. In conclusion, our results reveal the molecular regulation mechanism of TRIs on SG formation and provide novel insights into the toxicological mechanisms of TRIs.