Background: The loss of PTEN function presents in up to 50% of late-stage prostate cancers, and is therefore a potential target for therapeutics. PTEN-deficient cells depend on de novo pyrimidine synthesis, a feature that can present a vulnerability. Methods: We utilized in vitro growth assays and in vivo xenograft models to test the effect of de novo pyrimidine synthesis inhibition on prostate cell lines. Results: Here, we demonstrate that PTEN-deficient prostate cancer cell lines are susceptible to inhibition of de novo pyrimidine synthesis by leflunomide. Tumor growth inhibition was observed in vitro and in vivo following leflunomide treatment, and is likely due to an overwhelming accumulation of DNA damage. Conclusions: Our work highlights that synthetic lethality arises upon the combination of PTEN loss and leflunomide treatment in prostate cancer, and may present a therapeutic opportunity for this patient population.