Tubular injury is closely correlated with the development of progressive diabetic nephropathy (DN), particularly in cases of type 2 diabetes. The apoptosis of tubular cells has been recognized as a major cause of tubular atrophy, followed by tubulointerstitial fibrosis. Electron transfer flavoprotein beta (ETF beta) is known as an important electron acceptor in energy metabolism, but its role in DN was not fully understood. In the present study, we examined the expression pattern of ETF beta using diabetic kidney samples and further investigated ETF beta involvement in tubular epithelial cell (TEC) apoptosis. Human renal biopsy specimens from patients with DN as well as a spontaneous rat model of diabetes using Otsuka Long-Evans Tokushima fatty (OLETF) rats, were employed in order to examine the expression of ETF and cell apoptosis in kidneys during the development of DN (for the rats, at 36 and 56 weeks of age respectively). Moreover, ETF beta siRNA was used to investigate the role of ETF beta in the apoptosis of renal tubular cells. Our present results showed that the expression of ETF beta in the kidneys was progressively decreased both in patients with DN and OLETF rats, which coincided with progressive renal injury and TEC apoptosis. In addition, the in vitro study demonstrated that knockdown of ETF beta caused apoptosis in tubular cells, as proven by the increased expression of pro-apoptotic proteins and TUNEL assay. Therefore, the findings of our present study suggest that ETF beta plays an important role in renal tubular cell apoptosis during the progression of DN.