Phosphatase and tensin homolog (PTEN), deleted on chromosome 10, is a potent tumor suppressor. PTEN expression is reduced in advanced bladder cancer and reduction correlates with disease stage. To gain insights into the function of PTEN in human bladder cancer by identifying its binding partners, we developed a novel IPTG inducible PTEN expression system and evaluated this system in the PTEN null UMUC-3 human bladder cancer xenograft model. In this model, induction of PTEN in vivo resulted in reduced tumor growth. We used mass spectrometry to identify PTEN interaction partners in these cells, which identified known interaction partners major vault protein (MVP) and paxillin as well as a novel interaction partner, TRK fused gene (TFG). In conclusion, using a biologically relevant model system to dissect PTEN tumor suppressor function in human bladder cancer, we identified three molecules important for many cellular functions in complex with PTEN.