Purpose: Although the ABCB1 (P-glycoprotein) drug transporter is a constituent of several blood-tissue barriers (i.e., blood-brain and blood-nerve), its participation in a putative blood-heart barrier has been poorly explored. ABCB1 could decrease the intracardiac concentrations of drugs that cause QT prolongation and cardiotoxicity. Experimental Design: ABCB1-related romidepsin transport kinetics were explored in LLC-PK1 cells transfected with different ABCB1 genetic variants. ABCB1 plasma and intracardiac concentrations were determined in Abcb1a/1b (-/-) mice and wild-type FVB controls. These same mice were used to evaluate romidepsin-induced heart rate-corrected QT interval (QTc) prolongation over time. Finally, a cohort of 83 individuals with available QTcB and ABCB1 genotyping data were used to compare allelic variation in ABCB1 versus QTc-prolongation phenotype. Results: Here, we show that mice lacking the ABCB1-type P-glycoprotein have higher intracardiac concentrations of a model ABCB1 substrate, romidepsin, that correspond to changes in QT prolongation from baseline (ΔQTc) over time. Consistent with this observation, we also show that patients carrying genetic variants that could raise ABCB1 expression in the cardiac endothelium have lower ΔQTc following a single dose of romidepsin. Conclusions: To our knowledge, this is the first evidence that Abcb1-type P-glycoprotein can limit intracardiac exposure to a drug that mediates QT prolongation and suggests that certain commonly inherited polymorphisms in ABCB1 may serve as markers for QT prolongation following the administration of ABCB1-substrate drugs.