Amino acid transporter ATB⁰/ASCT2 is responsible for most glutamine uptake in human hepatoma cells. As this transporter is not expressed in normal hepatocytes, we hypothesized that its expression is necessary for growth of human liver cancer cells. To test this hypothesis, SK-Hep cells were stably transfected with an inducible 1.3 kb ATB⁰/ASCT2 antisense RNA expression plasmid under the transcriptional control of mifepristone (MFP), a synthetic steroid. Induced antisense RNA expression in monolayer cultures decreased ATB⁰/ASCT2 mRNA levels by 73% and glutamine transport rates by 65% compared to controls after 24 h, leading to a 98% decrease in cell number after 48 h. Cellular death was attributable to apoptosis based upon cellular blebbing, caspase-3 activation, vital dye and TUNEL staining and PARP cleavage. Transporter knockdown also markedly increased caspase 2 and 9 activities, marginally enhanced caspase 8 activity and dramatically increased ASCT1 mRNA levels, presumably as a futile compensatory response. Apoptosis elicited via transporter silencing was not attributable to the double stranded RNA-dependent protein kinase (PKR) pathway. For comparison, glutamine deprivation also caused apoptotic cell death but with slower temporal kinetics, stimulated caspase 2 and 3 but not caspase 8 or 9 activities, and led to considerable PARP cleavage. Thus, ASCT2 suppression exerts pro-apoptotic effects transcending those of glutamine starvation alone. We conclude that ATB⁰/ASCT2 expression is necessary for SK-Hep growth and viability, and suggest that it be further explored as a selective target for human hepatocellular carcinoma.