Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits anti-neoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. . There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here we show that serine withdrawal increases the anti-neoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation attenuated the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, dietary restriction of serine reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as agents that target blunt cancer cell energy metabolism and tumor growth.