PRMT5 is a therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer. CDK4/6 inhibitors (CDK4/6i) improve survival in ER+ breast cancer, but resistance develops, leaving patients with limited options. RB1 loss-of-function contributes to resistance, but effective therapies are unclear. A genome-wide CRISPR screen identified PRMT5 as a vulnerability in ER+/RB-deficient cells. PRMT5 inhibition blocks the G1-to-S cell cycle transition, leading to growth arrest. Proteomics revealed FUS as a downstream effector of PRMT5. Inhibition of PRMT5 dissociates FUS from RNA polymerase II, causing hyperphosphorylation of Ser2 in RNA polymerase II, intron retention, and downregulation of DNA synthesis proteins. PRMT5 inhibitor pemametostat and ER degrader fulvestrant synergistically inhibit growth of ER+/RB-deficient xenografts. These findings suggest dual ER and PRMT5 blockade as a potential strategy to overcome CDK4/6i resistance in ER+/RB-deficient breast cancer. PRMT5 is a key enzyme in arginine methylation, involved in epigenetic regulation, RNA processing, and cell cycle progression. PRMT5 inhibition blocks G1-to-S transition in RB-deficient cells, independent of RB. FUS is a functional substrate of PRMT5, and its dissociation from RNA polymerase II leads to hyperphosphorylation of Ser2 and intron retention, affecting DNA synthesis. PRMT5 inhibition also results in hyperphosphorylation of Ser2 Pol II and dysregulation of RNA splicing, leading to intron retention in cell cycle-related genes. PRMT5 inhibition synergizes with ER antagonists to block G1-to-S transition in ER+/RB-deficient breast cancer. These findings highlight PRMT5 as a therapeutic target for ER+/RB-deficient breast cancer.PRMT5 is a therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer. CDK4/6 inhibitors (CDK4/6i) improve survival in ER+ breast cancer, but resistance develops, leaving patients with limited options. RB1 loss-of-function contributes to resistance, but effective therapies are unclear. A genome-wide CRISPR screen identified PRMT5 as a vulnerability in ER+/RB-deficient cells. PRMT5 inhibition blocks the G1-to-S cell cycle transition, leading to growth arrest. Proteomics revealed FUS as a downstream effector of PRMT5. Inhibition of PRMT5 dissociates FUS from RNA polymerase II, causing hyperphosphorylation of Ser2 in RNA polymerase II, intron retention, and downregulation of DNA synthesis proteins. PRMT5 inhibitor pemametostat and ER degrader fulvestrant synergistically inhibit growth of ER+/RB-deficient xenografts. These findings suggest dual ER and PRMT5 blockade as a potential strategy to overcome CDK4/6i resistance in ER+/RB-deficient breast cancer. PRMT5 is a key enzyme in arginine methylation, involved in epigenetic regulation, RNA processing, and cell cycle progression. PRMT5 inhibition blocks G1-to-S transition in RB-deficient cells, independent of RB. FUS is a functional substrate of PRMT5, and its dissociation from RNA polymerase II leads to hyperphosphorylation of Ser2 and intron retention, affecting DNA synthesis. PRMT5 inhibition also results in hyperphosphorylation of Ser2 Pol II and dysregulation of RNA splicing, leading to intron retention in cell cycle-related genes. PRMT5 inhibition synergizes with ER antagonists to block G1-to-S transition in ER+/RB-deficient breast cancer. These findings highlight PRMT5 as a therapeutic target for ER+/RB-deficient breast cancer.