A large-panel gene expression analysis was conducted to identify biomarkers associated with the effectiveness of adding palbociclib to fulvestrant. The PALOMA-3 ( ClinicalTrials.gov identifier: NCT01942135) trial randomly assigned 521 endocrine-pretreated patients with metastatic breast cancer to receive palbociclib plus fulvestrant or placebo plus fulvestrant. Primary analysis was first conducted on 10 genes on the basis of pathway biology and evidence from previous studies followed by a systematic panel-wide search among 2,534 cancer-related genes. The association of gene expression with the effect of palbociclib on progression-free survival (PFS) was evaluated using Cox proportional hazards regression analysis, with gene expression as a continuous variable or dichotomized by median. An independent breast cancer cohort from the Preoperative Palbociclib (POP) Clinical Trial ( ClinicalTrials.gov identifier: NCT02008734) was used for validation, in 61 patients with primary breast cancer treated with 2 weeks of palbociclib. In the PALOMA-3 trial, 302 patients had tumor tissue analyzed (palbociclib arm, 194 patients; placebo arm, 108 patients). Palbociclib efficacy was lower in patients with high versus low cyclin E1 ( ) mRNA expression (median PFS: palbociclib arm, 7.6 14.1 months; placebo arm, 4.0 4.8 months, respectively; interaction unadjusted = .00238; false discovery rate-adjusted = .0238). mRNA was more predictive in metastatic than in archival primary biopsy tissue samples. No significant interaction was found between treatment and expression levels of CDK4, CDK6, cyclin D1, and RB1. Palbociclib was efficacious in both luminal A and luminal B tumors. High mRNA expression was associated with poor antiproliferative activity of palbociclib in the POP trial ( = .005). Addition of palbociclib to fulvestrant demonstrated efficacy in all biomarker groups, although high mRNA expression was associated with relative resistance to palbociclib.