T-2 toxin, one member of the type A trichothecene family, induces the apoptosis of human hepatocytes (L02) and murine Leydig cells (TM3), as well as mitochondrial dysfunctions. In the present study, we attempted to investigate whether T-2 toxin toxicity is related to mitochondrial dysfunction and mitophagy. We found that T-2 toxin might induce autophagy and mitophagy in TM3 cells (TM3) in a concentration-dependent manner. In addition, T-2 toxin could induce mitochondrial dysfunction, depolarization, and fission concentration-dependently. The inducible effects of T-2 toxin on mitophagy, mitochondrial dysfunction, and cell apoptosis could all be significantly reversed by autophagy inhibitor, 3 MA. Finally, DRP-1 participated in the inducible effects of T-2 toxin on TM3 cell mitophagy, mitochondrial dysfunction, and cell apoptosis. In summary, mitophagy and mitochondrial dysfunction are essential mechanisms of the toxicity induced by T-2 toxin. Thus, our findings provide a rationale for further studies on selectively targeting mitophagy to improve mitochondrial dysfunction and to protect cells from T-2 toxin-induced toxicity.