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Abstract Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) have been approved in combination with endocrine therapy (ET) to treat estrogen receptor-positive (ER+) metastatic breast cancer (BC). However, drug resistance represents the leading cause of breast cancer patients mortality. This study aimed to identify novel resistance mechanisms to ER antagonists in combination with CDK4/6 inhibitors. We generated two ER+ BC cell lines, T47D and MCF7, resistant to the combination of the ER antagonist fulvestrant and CDK4/6i abemaciclib, named T47D-FAR and MCF7-FAR. Transcriptomic analysis revealed common up-regulation of genes involved in MAPK and epithelial to mesenchymal transition (EMT) pathways in FAR cells, sustaining their hyper-invasive phenotype and increased anchorage-independent growth, compared to sensitive cells. FAR cells showed higher p21-activated kinase 1 (Pak1) expression and phosphorylation levels than parental cells. PAK1 knockdown by siRNAs hampered cell proliferation, reduced anchorage-independent growth and invasive properties of T47D-FAR and MCF7-FAR, re-sensitizing them to fulvestrant and abemaciclib. Conversely, over-expression of PAK1 in MCF7 and T47D cells increased tumor spheroids’ growth and invasion and reduced sensitivity to fulvestrant and abemaciclib, confirming its role in inducing drug resistance. Finally, treatment with Pak1 inhibitors, PF-3758309 (PF309) and NVS-PAK1-1, restored cell sensitivity to fulvestrant and abemaciclib of MCF7-FAR and T47D-FAR cells, both in vitro and in vivo. In conclusion, our data suggested a pivotal role for Pak1 in resistance to ET and CDK4/6i in ER+ breast cancers. These data might promote the rationale for the development of novel Pak1 inhibitors for treatment of patients with ER+ BC progressing on ET plus CDK4/6i.