OBJECTIVE To investigate the effects of adenovirus-mediated inducible nitric oxide synthase gene transfection on bladder transitional cell carcinoma T24 cells, and to provide novel insights and approaches to clinical therapies against bladder transitional cell carcinoma. METHODS Firstly, construct recombinant adenovirus vector pAd-iNOS of iNOS, followed by transfection of pAd-iNOS into HECK293 packaging cells. Thirdly, harvest recombinant adenovirus rAd-iNOS after amplification and purification procedures. Finally, transfect the recombinant adenovirus rAd-iNOS into human bladder carcinoma T24 cells and examine the effect of rAd-iNOS transfection on apoptosis of T24 and possible mechanism. RESULTS As shown by this study, the recombinant adenovirus rAd-iNOS was constructed successfully. The virus titer was 5.8×10(8) PFU/mL and recombinant was verified by PCR analysis. Transfection of adenovirus rAd-iNOS into T24 cells could induce secretion of high NO concentration, P53 protein expression up-regulation, as well as promotion of T24 cell apoptosis. CONCLUSIONS The transfection of human bladder carcinoma T24 cells from recombinant adenovirus rAd-iNOS was confirmed to induce intracellular iNOS over-expression, high production of NO, up-regulation of intracellular P53 expression and promotion of cell apoptosis.