Mycobacterium bovis Bacillus Calmette-Guérin (BCG) use in the treatment of bladder cancer was first reported in 1976, but the mechanism of the induced antitumor activity has still not been fully explained. BCG is a potent immunostimulant, normally producing a Th1 cytokine response, including IFN. Recent studies have shown CpG oligodeoxynucleotide induce tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression via IFN production. Given that Mycobacterial DNA contains high amounts of CpG motifs, we hypothesized that BCG’s antitumor properties are akin to CpG oligodeoxynucleotide, where the cytokine response to BCG induces TRAIL up-regulation. Using ELISA, urine IFN-γ, and TRAIL levels were initially undetectable in BCG therapy patients but were high after later induction treatments. More importantly, patients that responded to BCG therapy had significantly higher urine TRAIL levels, which killed bladder tumor cells in vitro versus nonresponders. Flow cytometry of fresh urine revealed TRAIL-expressing neutrophils. Given these data, we propose TRAIL plays a role in BCG-induced antitumor effects.