Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate high glucose (HG)-induced podocyte apoptosis remain poorly understood. Conditionally immortalized mouse podocytes were cultured in HG medium. A chemical inhibitor or a specific short-hairpin RNA (shRNA) vector was used to inhibit the activation of the Notch pathway and the PI3K/Akt pathway in HG-treated podocytes. Western blotting and real-time PCR were used to evaluate the levels of Notch, PI3K/Akt, and apoptotic pathway signaling. The apoptosis rate of HG-treated podocytes was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling and annexin V/propidium iodide staining. In HG-treated podocytes, PI3K/Akt pathway activation prevented podocyte apoptosis in the early stage of HG stimulation and Notch pathway-induced podocyte apoptosis in the late stage of HG stimulation. The inhibition of the Notch pathway or the activation of the PI3K/Akt pathway prevented cell apoptosis in HG-treated podocytes. These findings suggest that the Notch and PI3K/Akt pathways may mediate HG-induced podocyte apoptosis.