Glioblastomas and brain metastases are highly proliferative brain tumors with short survival times. Previously, using ¹³C-NMR analysis of brain tumors resected from patients during infusion of ¹³C-glucose, we demonstrated that there is robust oxidation of glucose in the citric acid cycle, yet glucose contributes less than 50% of the carbons to the acetyl-CoA pool. Here, we show that primary and metastatic mouse orthotopic brain tumors have the capacity to oxidize [1,2-¹³C]acetate and can do so while simultaneously oxidizing [1,6-¹³C]glucose. The tumors do not oxidize [U-¹³C]glutamine. In vivo oxidation of [1,2-¹³C]acetate was validated in brain tumor patients and was correlated with expression of acetyl-CoA synthetase enzyme 2, ACSS2. Together, the data demonstrate a strikingly common metabolic phenotype in diverse brain tumors that includes the ability to oxidize acetate in the citric acid cycle. This adaptation may be important for meeting the high biosynthetic and bioenergetic demands of malignant growth.