The metabolism of branched-chain amino acids (BCAA) has recently been implicated in the growth of several cancer cell types. Gabapentin, a synthetic amino acid, is commonly used in high concentrations in this context to inhibit the cytosolic branched-chain amino acid transferase (BCAT1) enzyme. Here, we report that 10 mM gabapentin reduces the growth of HCT116 cells, which have an active branched-chain amino acid transferase but express very low levels of BCAT1, and presumably rely on the mitochondrial BCAT2 enzyme. Gabapentin did not affect transamination of BCAA to branched-chain keto acids (BCKA) in HCT116 cells, nor the reverse formation of BCAA from BCKA, indicating that the branched-chain amino acid transaminase is not inhibited. Moreover, the growth-inhibitory effect of gabapentin could not be rescued by supplementation with BCKA, and this was not due to the lack of uptake of BCKA, indicating that other effects of gabapentin are important. An untargeted LC-MS analysis of gabapentin-treated cells revealed a marked depletion of branched-chain carnitines. These results demonstrate that gabapentin at high concentrations can inhibit cell proliferation without affecting BCAT1 and may affect mitochondrial BCKA catabolism.