AbstractNAD[P]H:quinone oxidoreductase 1 (NQO1) regulates cell fate decisions in response to stress. Oxidative stress supports cancer maintenance and progression. Previously we showed that knockdown of NQO1 (NQO1^low) prostate cancer cells upregulate pro-inflammatory cytokines and survival under hormone-deprived conditions. Here, we tested the ability of NQO1^low cells to form tumors. We found NQO1^low cells form aggressive tumors compared with NQO1^high cells. Biopsy specimens and circulating tumor cells showed biochemical recurrent prostate cancer was associated with low NQO1. NQO1 silencing was sufficient to induce SMAD-mediated TGFβ signaling and mesenchymal markers. TGFβ treatment decreased NQO1 levels and induced molecular changes similar to NQO1 knockdown cells. Functionally, NQO1 depletion increased migration and sensitivity to oxidative stress. Collectively, this work reveals a possible new gatekeeper role for NQO1 in counteracting cellular plasticity in prostate cancer cells. Further, combining NQO1 with TGFβ signaling molecules may serve as a better signature to predict biochemical recurrence.