Nuclear receptor subfamily 4 group A member 1 (NR4A1)‑induced chondrocyte death plays a critical role in the development of osteoarthritis through poorly defined mechanisms. The present study aimed to investigate the role of NR4A1 in regulating chondrocyte death in response to tumor necrosis factor‑α (TNF‑α) and cycloheximide (CHX) treatment, with a focus on mitochondrial fission and the AMP‑activated protein kinase (AMPK) signaling pathway. The results demonstrated that NR4A1 was significantly upregulated in TNF‑α and CHX exposed chondrocytes. Increased NR4A1 triggered mitochondrial fission via the AMPK/dynamin‑related protein 1 (Drp1) pathway, resulting in mitochondrial dysfunction, and mitochondrial permeability transition pore (mPTP) opening‑related cell death. Furthermore, excessive mitochondrial fission impaired chondrocyte migration through imbalance of F‑actin homeostasis. Inhibiting NR4A1 attenuated TNF‑α and CHX‑induced mitochondrial fission and, thus, reduced mitochondrial dysfunction in chondrocytes, mPTP opening‑related cell death and migration injury. Altogether, the present data confirmed that mitochondrial fission was involved in NR4A1‑mediated chondrocyte injury via regulation of mitochondrial dysfunction, mPTP opening‑induced cell death and F‑actin‑related migratory inhibition.