Chordoma is a rare malignancy arising from the nucleus pulposus with poor long term outcomes. Chordoma predominantly occur in the sacrum and clivus but can also develop in the mobile spine. Clival chordoma can expand into the brain while sacral chordoma can migrate into the spinal cord. Chordoma is typically chemoresistant and is treated by radiation or surgical resection. Chordoma cells as well as the cells of the nucleus pulposus and the notochord, demonstrate a unique cytological diagnostic trait - large mucin and glycogen-filled vesicles termed physaliferous vacuoles. The formation of physaliferous vacuoles is poorly understood. Interrogation of chordoma gene signature data sets identified the solute carrier SLC6A12 as a candidate for regulation of physaliferous vacuoles. SLC6A12 transports the osmolyte amino acids GABA and betaine across membranes including vacuolar membranes. SLC6A12 protein expression in three chordoma cell lines was demonstrated by confocal immunofluorescent microscopy with protein expression found in the cytoplasm. Chordoma cells treated with GABA transporter inhibitor (S)-SNAP-5114, showed reduced cell size, loss of SLC6A12 protein expression and loss of physaliferous vacuoles. (S)-SNAP-5114 induced apoptosis in chordoma cell lines. These data suggest that the physaliferous vacuoles sequester amounts of GABA and/or betaine. Blocking the transport of these amino acids may lead loss of osmotic regulation, to a toxic build-up of GABA or betaine in the cytosol and apoptosis. Taken together, physaliferous vacuoles could serve as a target for developing novel therapeutics to treat chordoma.