Digitalis-like compounds (DLCs), such as digoxin and digitoxin that are derived from digitalis species, are currently used to treat heart failure and atrial fibrillation, but have a narrow therapeutic index. Drug-drug interactions at the transporter level are frequent causes of DLCs toxicity. P-glycoprotein (P-gp, ABCB1) is the primary transporter of digoxin and its inhibitors influence pharmacokinetics and disposition of digoxin in the human body; however, the involvement of P-gp in the disposition of other DLCs is currently unknown. In present study, the transport of fourteen DLCs by human P-gp was studied using membrane vesicles originating from human embryonic kidney (HEK293) cells overexpressing P-gp. DLCs were quantified by liquid chromatography-mass spectrometry (LC-MS). The Lily of the Valley toxin, convallatoxin, was identified as a P-gp substrate (Km: 1.1±0.2 mM) in the vesicular assay. Transport of convallatoxin by P-gp was confirmed in rat in vivo, in which co-administration with the P-gp inhibitor elacridar, resulted in increased concentrations in brain and kidney cortex. To address the interaction of convallatoxin with P-gp on a molecular level, the effect of nine alanine mutations was compared with the substrate N-methyl quinidine (NMQ). Phe343 appeared to be more important for transport of NMQ than convallatoxin, while Val982 was particularly relevant for convallatoxin transport. We identified convallatoxin as a new P-gp substrate and recognized Val982 as an important amino acid involved in its transport. These results contribute to a better understanding of the interaction of DLCs with P-gp.