It has been reported that sodium fluoride (NaF) suppresses the proliferation and induces apoptosis of chondrocytes. However, the cellular and molecular mechanisms of the effect have not been elucidated. Therefore, the aim of this study was to evaluate the mechanisms of the effects of NaF on primary cultured rat chondrocytes in vitro. Chondrocytes were treated with NaF at concentrations of 0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mM. Cell viability decreased and the rate of apoptotic cells increased significantly with the gradient concentration of NaF in a time- and dose-dependent manner. Electron microscopy revealed cytoplasmic, organelle and nuclear alterations in the ultrastructure of chondrocytes exposed to various NaF concentrations. The cell cycle distribution was analyzed by flow cytometry, and the results indicated that NaF induced G2 cell cycle arrest. Western blotting was used to detect the apoptotic pathways. Downregulation of the Bcl-2 protein and upregulation of Bax, cleaved caspase-9, -12 and -3 proteins suggested that NaF was capable of inducing apoptosis through the mitochondrial and endoplasmic reticulum pathways. The results also showed that the levels of hypoxia-inducible factor 1α (HIF-1α), sex determining region Y box gene 9 (Sox9) and the collagen II (Col II) protein of the NaF groups were lower compared to those of the control groups. Thus, NaF may induce apoptosis through the downregulation of HIF-1α and disrupt the synthesis of extracellular matrix (ECM) through the downregulation of HIF-1α via the Sox9 pathway in primary cultured rat chondrocytes.