Purpose: Resistance to endocrine therapy in estrogen receptor-positive (ER +) breast cancer remains a major clinical problem. Recently, the CDK4/6 inhibitor palbociclib combined with letrozole or fulvestrant was approved for treatment of ER + advanced breast cancer. However, the role of CDK4/6 in endocrine resistance and their potential as predictive biomarkers of endocrine treatment response remains undefined. Experimental Design: We investigated the specific role of increased CDK6 expression in fulvestrant-resistant cells by gene knockdown and treatment with palbociclib, and evaluated the effect in cell proliferation, apoptosis, and kinase activity. Furthermore, we evaluated CDK6 expression in metastatic samples from breast cancer patients treated or not with fulvestrant. Results: We found increased expression of CDK6 in two fulvestrant-resistant cell models versus sensitive cells. Reduction of CDK6 expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib resensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer patients treated with fulvestrant (N = 45 and 46) correlated significantly with shorter progression-free survival (PFS) on fulvestrant treatment (P = 0.0006 and 0.018), whereas no association was observed in patients receiving other first- or second-/third-line endocrine treatments (N = 68, P = 0.135 and 0.511, respectively). Conclusions: Our results indicate that upregulation of CDK6 may be an important mechanism in overcoming fulvestrant-mediated growth inhibition in breast cancer cells. Patients with advanced ER + breast cancer exhibiting high CDK6 expression in the metastatic lesions show shorter PFS upon fulvestrant treatment and thus may benefit from the addition of CDK4/6 inhibitors in their therapeutic regimens.