l-ascorbate (l-ascorbic acid, vitamin C) clearly has an inhibitory effect on cancer cells. However, the mechanism underlying differential sensitivity of cancer cells from same tissue to l-ascorbate is yet to be clarified. Here, we demonstrate that l-ascorbate has a selective killing effect, which is influenced by sodium-dependent vitamin C transporter 2 (SVCT-2) in human breast cancer cells. Treatment of human breast cancer cells with l-ascorbate differentially induced cell death, dependent on the SVCT-2 protein level. Moreover, knockdown of endogenous SVCT-2 via RNA interference in breast cancer cells expressing high levels of the protein induced resistance to l-ascorbate treatment, whereas transfection with SVCT-2 expression plasmids led to enhanced l-ascorbate chemosensitivity. Surprisingly, tumor regression by l-ascorbate administration in mice bearing tumor cell xenograft also corresponded to the SVCT-2 protein level. Interestingly, SVCT-2 expression was absent or weak in normal tissues, but strongly detected in tumor samples obtained from breast cancer patients. In addition, enhanced chemosensitivity to l-ascorbate occurred as a result of caspase-independent autophagy, which was mediated by beclin-1 and LC3 II. In addition, treatment with N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, suppressed the induction of beclin-1 and LC3 II, implying that the differential SVCT-2 protein-dependent l-ascorbate uptake was attributable to intracellular ROS induced by l-ascorbate, subsequently leading to autophagy. These results suggest that functional SVCT-2 sensitizes breast cancer cells to autophagic damage by increasing the l-ascorbate concentration and intracellular ROS production and furthermore, SVCT-2 in breast cancer may act as an indicator for commencing l-ascorbate treatment.