In order to determine whether the glucose-alanine cycle regulates rates of hepatic mitochondrial oxidation in humans, we applied positional isotopomer NMR tracer analysis (PINTA) to assess rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in healthy volunteers following both an overnight (12 hours) and a 60-hour fast. Following the 60-hour fast, rates of endogenous glucose production and mitochondrial oxidation decreased, whereas rates of hepatic pyruvate carboxylase flux remained unchanged. These reductions were associated with reduced rates of alanine turnover, assessed by [3-¹³C]alanine, in a subgroup of participants under similar fasting conditions. In order to determine whether this reduction in alanine turnover was responsible for the reduced rates of hepatic mitochondrial oxidation, we infused unlabeled alanine into another subgroup of 60-hour fasted subjects to increase rates of alanine turnover, similar to what was measured after a 12-hour fast, and found that this perturbation increased rates of hepatic mitochondrial oxidation. Taken together, these studies demonstrate that 60 hours of starvation induce marked reductions in rates of hepatic mitochondrial oxidation, which in turn can be attributed to reduced rates of glucose-alanine cycling, and reveal a heretofore undescribed role for glucose-alanine in the regulation of hepatic mitochondrial oxidation in humans.