Cancer cells use precursors derived from tricarboxylic acid (TCA) cycle to support their unlimited growth. However, continuous export of TCA cycle intermediates results in the defect of mitochondrial integrity. Mitochondria glutamine metabolism plays an essential role for the maintenance of mitochondrial functions and its biosynthetic roles by refilling the mitochondrial carbon pool. Here we report that human pancreatic ductal adenocarcinoma (PDAC) cells have a distinct dependence on mitochondrial glutamine metabolism. Whereas glutamine flux into mitochondria contributes to proliferation of most cancer cells, enhanced glutamine anaplerosis results in a pronounced suppression of PDAC growth. A cell membrane permeable α-ketoglutarate analog or overexpression of glutamate dehydrogenase lead to decreased proliferation and increased apoptotic cell death in PDAC cells but not other cancer cells. We found that enhanced glutamine anaplerosis inhibits autophagy, required for tumorigenic growth of PDAC, by activating mammalian TORC1. Together, our results reveal that glutamine anaplerosis is a crucial regulator of growth and survival of PDAC cells, which may provide novel therapeutic approaches to treat these cancers.