Hyperglycemia promotes podocyte apoptosis and contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms of hyperglycemia‑induced podocyte apoptosis remain unknown. Recent studies have implicated Src‑associated substrate during mitosis of 68 kDa (Sam68) in various cellular processes including RNA metabolism, apoptosis, signal transduction. This study sought to examine the effect of Sam68 on high glucose (HG)‑induced podocytes apoptosis, and the mechanism underlying this effect. Immortalized mouse podocytes were exposed to medium containing normal glucose, or HG and Sam68 siRNA, respectively. The expression of Sam68 in podocytes was determined by fluorescence quantitative PCR (qPCR), immunofluorescence and immunoblotting. The role of Sam68 in HG‑induced podocyte apoptosis was further evaluated by inhibiting Sam68 expression by Sam68 siRNA and performing flow cytometry. The mRNA and protein expression of pro‑apoptosis gene Bax and anti‑apoptotic gene Bcl‑2 were assessed by qRCR and immunoblotting. In the present study, it was first demonstrated that Sam68 was upregulated in a time and dose‑dependent manner in in vitro HG‑treated podocytes. Pretreatment with Sam68 siRNA markedly decreased nuclear Sam68 expression. Moreover, the effects of HG‑induced apoptosis were also abrogated by Sam68 knockdown in cultured podocytes. Furthermore, HG increased Bax and decreased Bcl‑2 protein expression in cultured podocytes, and this effect was blocked by Sam68 knockdown. The results of the present study revealed that Sam68 mediated HG‑induced podocyte apoptosis, probably through the Bax/Bcl‑2 signaling pathway, and thus may be a potential therapeutic target for DN.