The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of hOCTN1 and hOCTN2 (a Na⁺-dependent carnitine carrier with Na⁺-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC organic cation transporters such as hOCT1 and EMT/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of ATB⁰⁺ (an amino acid transporter with a Na⁺/Cl^--dependent carnitine transport activity), and Flipt2/OCT6 (a splice variant of CT2: a testis-specific Na⁺-dependent canitine transporter). TEA uptake was pH-dependent. Carnitine uptake was dependent on sodium, and partly on chloride, compatible with hOCTN2, and ATB⁰⁺ function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by Km of 5.1 µM for carnitine and 1.6 mM for TEA, respectively; apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake, but required supra-therapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotics challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/nutrient transporters in human mammary gland drug transfer.