Regulatory T cells (Tregs) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral Treg stability and function but is dispensable for peripheral immune tolerance. Treg-restricted Nrp1 deletion results in profound tumor resistance due to Treg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of Treg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1+ Tregs correlates with poor prognosis in melanoma and head and neck squamous cell carcinoma. Using a mouse model of melanoma where Nrp1-deficient (Nrp1–/–) and wild-type (Nrp1+/+) Tregs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1–/– Tregs produce interferon-γ (IFNγ), which drives the fragility of surrounding wild-type Tregs, boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFNγ-induced Treg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting Treg fragility may be efficacious. Display omitted •Increased percentage of human NRP1+ intratumoral Tregs correlates with poor prognosis•Nrp1-deficient Tregs undermine the function of wild-type Tregs via IFNγ•Hypoxia may drive IFNγ-induced Treg fragility via Hif1α in the tumor microenvironment•IFNγ-mediated Treg functional fragility is required for response to PD1 blockade Driving Treg fragility in the tumor microenvironment is critical for the efficacy of cancer checkpoint blockade therapy.