Bladder cancer is currently the sixth most common cancer in the United States. Treatments for invasive disease include surgical cystectomy and organ preservation with chemoradiation with suboptimal results and significant acute and chronic toxicity. Alternative therapies that address specific pathways leading to bladder cancer growth and invasion might provide better local/regional control and reduce distant spread. To this end, silibinin, a naturally derived flavonoid from milk-thistle, has been shown to arrest cell growth, have purported antioxidant properties, and cause apoptosis in human bladder transitional cell carcinoma cells. Silibinin has also been shown to markedly increase UVB-induced apoptosis in mouse epithelial-JB6 cells, up-regulating proapoptotic pathways. Determine the efficacy and optimal in vitro dosing of silibinin in combination with single-fraction, ionizing radiation in the treatment of bladder cancer. Six human bladder cancer cell lines (3HTB, HT1376, J82, RT4, TCC-SUP, and T24) were used, representing different grades and histologic types of bladder cancer. The silibinin doses examined ranged from 20 to 160 μM and the radiation doses from 0 to 6 Gray (Gy) at 60 keV. The silibinin was given concurrently to radiation exposure as well as 2 and 24 hours pre- and post-radiation treatment. Cell viability was determined 96 hours from the time of radiation treatment using a tetrazolium-based colorimetric assay. Individually, silibinin and radiation decreased cell viability in a dose- and time-dependent manner. At silibinin doses of 40 to 80 μM the effect of the silibinin and radiation was mildly additive when given concurrently. Considering the antioxidant properties of silibinin it was hypothesized that timing of the exposure of silibinin in relation to radiation was important to its efficacy. At time points when silibinin was added prior to radiation, results were inconclusive, perhaps due to the mixed effects of protection and additive toxicity. At time points when silibinin was added after radiation, a pattern suggestive of increased toxicity was observed, consistent with the known proapoptotic action of silibinin. These data suggest that single-agent silibinin inhibits cell viability in bladder cancer cells at high, sustained doses. Furthermore, this work suggests that silibinin may be an effective adjuvant to radiation when given after radiotherapy. Future studies in this area should focus on longer-duration experiments, fractionated radiation, the use of clonogenic assays to better characterize the radiation effect, and additional time points to establish the most efficacious treatment approaches.