Background <p>Palbociclib is the more extensively evaluated CDK4-6 inhibitor in term of safety, efficacy and sources of pharmacokinetic variability. However, real-life data on the correlation between palbociclib plasma concentration, drug-drug interactions, gene polymorphisms and efficacy remain scarce. We previously characterized the effects of co-medications (CYP3A4 and P-glycoprotein inhibitors and antacid drugs) on palbociclib plasma concentration, and here we wanted to identify factors that influence progression-free survival in the same cohort of patients.</p> Methods <p>This multicentric prospective clinical trial included patients with metastatic breast cancer treated with first-line palbociclib and an aromatase inhibitor. Efficacy (progression-free survival) and safety (high-grade neutropenia during the first two cycles) were reported, and the influence of covariates, such as drug-drug interactions, palbociclib plasma concentration and specific gene variants, were analyzed.</p> Results <p>In the 58 included patients, drug-drug interactions (antacid drugs) and pregnane X receptor (<i>NR1I2</i>) single nucleotide polymorphisms (SNP) were identified as survival prognostic factors, but not palbociclib concentration and high-grade neutropenia (<i>p</i> = 0.4 and <i>p</i> = 0.5). Concomitant antacid treatment reduced progression-free survival (26.2 vs. 32.6 months, <i>p</i> = 0.059) and the <i>NR1I2 rs10934498</i> and <i>rs2276707</i> SNPs affected survival (<i>p</i> = 0.002 and 0.004).</p> Conclusion <p>This study consolidated the data on palbociclib as first-line metastatic breast cancer treatment and identified factors that negatively influence survival (co-treatment with antacid drugs and <i>NR1I2</i> SNPs).</p> Trial registration <p>NCT04025541, Registration Date 2019-07-16.</p>