Inactivation of theRB1tumor suppressor gene is common in several types of therapy-resistant cancers, including metastatic castration-resistant prostate cancer, and predicts poor clinical outcomes. Effective therapeutic strategies againstRB1-deficient cancers remain elusive. Here, we showed thatRB1loss/E2F activation sensitized cancer cells to ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, by upregulating expression ofACSL4and enriching ACSL4-dependent arachidonic acid–containing phospholipids, which are key components of ferroptosis execution.ACSL4appeared to be a direct E2F target gene and was critical toRB1loss–induced sensitization to ferroptosis. Importantly, using cell line–derived xenografts and genetically engineered tumor models, we demonstrated that induction of ferroptosis in vivo by JKE-1674, a highly selective and stable GPX4 inhibitor, blockedRB1-deficient prostate tumor growth and metastasis and led to improved survival of the mice. Thus, our findings uncover an RB/E2F/ACSL4 molecular axis that governs ferroptosis and also suggest a promising approach for the treatment ofRB1-deficient malignancies.