Increased glutamine metabolism is a hallmark of cancer. Mitochondrial glutamic pyruvate transaminase (GPT2) catalyzes the reversible transamination between alanine and α-ketoglutarate (α-KG), also known as 2-oxoglutarate, to generate pyruvate and glutamate during cellular glutamine catabolism. However, the precise role of GPT2 in tumorigenesis remains elusive. Here, we report that in breast cancer tissue samples and breast cancer cell lines, GPT2 expression level was markedly elevated and correlated with the pathological grades of breast cancers. GPT2 overexpression increased the subpopulation of breast cancer stem cells in vitro and promoted tumorigenesis in mice. GPT2 reduced α-KG level in cells leading to the inhibition of proline hydroxylase 2 (PHD2) activity involved in the regulation of HIF1α stability. Accumulation of HIF1α, resulting from GPT2-α-KG-PHD2 axial, constitutively activates sonic hedgehog (Shh) signaling pathway. Overall, GPT2 promotes tumorigenesis and stemness of breast cancer cells by activating the Shh signaling, suggesting that GTP2 is a potential target for breast cancer therapy.