Notch signaling regulates cartilage formation and homeostasis. Kashin-Beck Disease (KBD), an endemic osteochondropathy, is characterized by severe cartilage degradation. The etiology of KBD is related to the exposure of HT-2 toxin, a mycotoxin and primary metabolite of T-2 toxin. This study aims to explore the role of HT-2 toxin in the Notch signaling regulation and extracellular matrix (ECM) metabolism of hiPSCs-Chondrocytes. Immunohistochemistry and qRT-PCR were employed to investigate the expression of Notch pathway molecules in KBD articular cartilage and primary chondrocytes. hiPSCs-Chondrocytes, derived from hiPSCs, were treated with 100 ng/mL HT-2 toxin and the γ-secretase inhibitor (DAPT) for 48h, respectively. The markers related to the Notch signaling pathway and ECM were assessed using qRT-PCR and Western blot. Notch pathway dysregulation was prominent in KBD cartilage. HT-2 toxin exposure caused cytotoxicity in hiPSCs-Chondrocytes, and activated Notch signaling by increasing the mRNA and protein levels of NOTCH1 and HES1. HT-2 toxin also upregulated ECM catabolic enzymes and downregulated ECM components (COL2A1 and ACAN), indicating ECM degradation. DAPT-mediated Notch signaling inhibition suppressed the mRNA and protein level of ADAMTS5 expression while enhancing ECM component expression in hiPSCs-Chondrocytes. This study suggests that HT-2 toxin may induce ECM degradation in hiPSCs-Chondrocytes through activating Notch signaling.