Glutamine is an essential nutrient that regulates energy production, redox homeostasis, and signaling in cancer cells. Despite the importance of glutamine in mitochondrial metabolism, the mitochondrial glutamine transporter has long been unknown. Here, we show that the SLC1A5 variant plays a critical role in cancer metabolic reprogramming by transporting glutamine into mitochondria. The SLC1A5 variant has an N-terminal targeting signal for mitochondrial localization. Hypoxia-induced gene expression of the SLC1A5 variant is mediated by HIF-2α. Overexpression of the SLC1A5 variant mediates glutamine-induced ATP production and glutathione synthesis and confers gemcitabine resistance to pancreatic cancer cells. SLC1A5 variant knockdown and overexpression alter cancer cell and tumor growth, supporting an oncogenic role. This work demonstrates that the SLC1A5 variant is a mitochondrial glutamine transporter for cancer metabolic reprogramming. Display omitted •The SLC1A5 variant is a mitochondrial glutamine transporter•The SLC1A5 variant has a mitochondrial targeting sequence•Hypoxia controls SLC1A5 variant expression through HIF-2α•The SLC1A5 variant mediates mitochondrial glutamine metabolism in cancer Despite the importance of glutamine in cancer metabolism, the mitochondrial glutamine transporter has long been unknown. Yoo et al. show that a variant of SLC1A5 has a mitochondrial targeting signal for mitochondrial localization and is induced by HIF-2α. SLC1A5 variant knockdown suppressed cancer cell growth, supporting an oncogenic role.