This study identifies protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in estrogen receptor-positive (ER+) breast cancer cells with RB1 loss-of-function alterations. Through a genome-wide CRISPR screen, PRMT5 was found to be essential for the survival of ER+/RB1-knockout (RBKO) cells, which are resistant to CDK4/6 inhibitors. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle, leading to growth arrest in RB1-knockout cells. Proteomics analysis reveals fused in sarcoma (FUS) as a downstream effector of PRMT5. Treatment with the PRMT5 inhibitor pemrametostat and the selective ER degrader fulvestrant synergistically inhibits the growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings suggest that dual blockade of ER and PRMT5 is a potential therapeutic strategy to overcome resistance to CDK4/6 inhibitors in ER+/RB-deficient breast cancer.This study identifies protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in estrogen receptor-positive (ER+) breast cancer cells with RB1 loss-of-function alterations. Through a genome-wide CRISPR screen, PRMT5 was found to be essential for the survival of ER+/RB1-knockout (RBKO) cells, which are resistant to CDK4/6 inhibitors. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle, leading to growth arrest in RB1-knockout cells. Proteomics analysis reveals fused in sarcoma (FUS) as a downstream effector of PRMT5. Treatment with the PRMT5 inhibitor pemrametostat and the selective ER degrader fulvestrant synergistically inhibits the growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings suggest that dual blockade of ER and PRMT5 is a potential therapeutic strategy to overcome resistance to CDK4/6 inhibitors in ER+/RB-deficient breast cancer.