Recently, 2-methoxyestradiol (2-ME) has been considered to be a potential anticancer agent but has not been investigated in bladder cancer. This study was conducted to clarify the role of 2-ME in bladder cancer cells. The bladder cancer cell line T-24 was treated with 2 μm 2-ME for 2 d. The T-24 cell viability, colony formation, invasion and apoptosis were observed in 2-ME-treated and control cells. The expression of hypoxia-inducible factor 1 alpha (HIF-1α) was detected using reverse transcription-polymerase chain reaction (RT-PCR). Then western blotting assay was applied to assess expressions of HIF-1α and apoptosis factors caspase-3 and Bcl-x proteins. The mRNA and protein expressions of HIF-1α in 2-ME-treated T-24 cells were remarkably lower than that of the control cells (P < 0.05). Treatment of 2-ME could significantly inhibit T-24 the cell viability, colony formation, invasion, and promote apoptosis (all P < 0.05). In addition, the protein expression of Caspase-3 was higher and that of Bcl-x protein was lower after administration of 2-ME compared to control (both P < 0.05). Collectively, we characterized the efficacy of 2-ME on bladder cancer T-24 cells as being mediated by inhibition of cell viability, colony fomation, invasion and promoting cell apoptosis, which may be achieved by suppressing HIF-1α levels. This study suggests 2-ME as a potential drug for bladder cancer therapy.