Inhibitor of apoptosis stimulatory protein phosphatase (iASPP) is a key inhibitor of p53 conserved from worm to human and is associated with cell proliferation and carcinogenesis in a variety of human cancers. Because iASPP is important for tumor cell apoptosis, it is a potential target for cancer gene therapy. However, it is still not clear whether iASPP is relevant to p53-deficient human bladder cancer. In the present study, iASPP was knocked down in bladder carcinoma 5637 and T24 cells (p53 defective) by lentiviral-mediated interfering short hairpin RNAs (siRNAs). MTT assay, BrdU incorporation assay, and colony formation assay were performed to investigate the role of iASPP on cell proliferation. It was suggested that iASPP knockdown led to cell growth deceleration and slow colony formation. A positive relationship between expression of iASPP and bladder cancer proliferation was found. The expression of iASPP may be critical for proliferation of bladder cancer cells. Our study indicates iASPP could be an important target for therapy in bladder cancer.