Bladder cancer is a common malignancy for which regional or metastatic disease is identified at diagnosis. The aim of this study was to determine whether tamoxifen (Tam), an estrogen receptor (ER) antagonist, can sensitize bladder cancer cell lines to gemcitabine (Gem) chemotherapy. ERα and ERβ protein levels were determined in each cell line using western blot analysis. The TCC-Sup, 5637, and RT4 bladder cancer cells were exposed to various concentrations and regimens of Tam or Gem alone or in combination. Cell viability and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide followed by flow cytometry. Apoptosis was then evaluated by western blot analysis. Treated TCC-Sup cells were subjected to soft agar colony formation assay to determine the cellular transformation. Western blot analysis results revealed ER expression in the three cell lines. TCC-Sup and 5637 cells treated with a combination of Tam and Gem had lower cell viabilities than those treated with Tam or Gem alone for 72 h in TCC-Sup and 5637. Compared with the other treatments, sequential Gem followed by Tam (Gem→Tam) treatment caused the largest increase in DNA fragmentation at 72 h in TCC-Sup cells. Western blot analysis results revealed that this sequential Gem→Tam treatment increased poly(ADP-ribose) polymerase cleavage in TCC-Sup cells. Sequential Gem→Tam inhibited the cell transformation in TCC-Sup cells. In conclusion, sequential Gem→Tam enhanced the cytotoxicity of Gem in vitro. This regimen be useful to enhance the efficacy of Gem in bladder cancer. However, future in vivo studies are required to verify the results.