Hypoxia-induced apoptosis of human periodontal ligament cells (hPDLCs) is an important contributor to the progression of various periodontal diseases. Although leptin has been shown to protect connective tissue cells against hypoxia-induced injury, whether it might do so by attenuating hypoxia-induced apoptosis in hPDLCs remains unclear. Here, using CoCl2 treatment, we simulated hypoxic conditions in hPDLCs and explored whether apoptosis and reactive oxygen species (ROS) levels were related to hypoxia. After small interfering RNA (siRNA) inhibition of leptin and hypoxia-inducible factor-1 alpha (HIF-1 alpha), the levels of apoptosis, ROS and leptin expression were measured. We showed that in CoCl2-treated hPDLCs, significantly higher cell apoptosis rates and ROS accumulation were observed. Cobalt chloride also increased leptin and HIF-1 alpha expression in hPDLCs. Further investigation of the pathway demonstrated that inhibition of ROS attenuated hypoxia-induced cell apoptosis and leptin expression, whereas siRNA inhibition of leptin aggravated hypoxia-induced cell apoptosis and ROS accumulation. Hypoxia induces cell apoptosis and leptin expression in hPDLCs through the induction of ROS and HIF-1 alpha pathways, and leptin shows feedback inhibition on ROS-mediated apoptosis in hPDLCs. These findings suggest a new application of leptin for hypoxic damage in periodontal diseases.