This study aimed to examine the effects of Lefty A protein on transforming growth factor-β1 (TGF-β1)-mediated apoptosis in human renal tubular epithelial cells (HK-2). HK-2 cells were transfected with the human Lefty gene to induce the secretion of endogenous Lefty A protein. Following exposure of the HK-2 cells to recombinant human TGF-β1 (10 ng/ml), p-Smad2/3 protein levels were examined by western blot analysis, and cellular apoptosis was detected by flow cytometry 6, 12, 24 and 48 h following TGF-β1 treatment. Coculture of renal tubular epithelial cells with TGF-β1 resulted in a significant increase in p-Smad2/3 protein levels and the rate of cell apoptosis, which were attenuated by liposome-mediated transfection with the Lefty gene. Lefty A protein was able to inhibit the TGF-β1/Smad signaling pathway and markedly attenuate TGF-β1-mediated apoptosis in human renal tubular epithelial cells. Taken together, these results indicated that the TGF-β1/Smad signaling pathway most likely mediates apoptosis in renal tubular epithelial cells. In addition, Lefty A protein is capable of inhibiting the TGF-β1/Smad pathway to reduce TGF-β1/Smad-mediated apoptosis in renal tubular epithelial cells. This study may provide novel insights into the prevention and treatment of urinary tract obstruction disease using Lefty A protein.