Retinoblastoma (Rb) protein is a tumor suppressor that is dysregulated in a majority of human cancers. Rb functions to inhibit cell cycle progression in part by directly disabling the E2F family of cell cycle-promoting transcription factors. Because the de novo synthesis of multiple glutamine-derived anabolic precursors is required for cell cycle progression, we hypothesized that Rb also may directly regulate proteins involved in glutamine metabolism. We examined glutamine metabolism in mouse embryonic fibroblasts (MEFs) isolated from mice that have triple knock-outs (TKO) of all three Rb family members (Rb-1, Rbl1 and Rbl2) and found that loss of global Rb function caused a marked increase in (13)C-glutamine uptake and incorporation into glutamate and tricarboxylic acid cycle (TCA) intermediates in part via upregulated expression of the glutamine transporter ASCT2 and the activity of glutaminase 1 (GLS1). The Rb-controlled transcription factor E2F-3 altered glutamine uptake by direct regulation of ASCT2 mRNA and protein expression, and E2F-3 was observed to associate with the ASCT2 promoter. We next examined the functional consequences of the observed increase in glutamine uptake and utilization and found that glutamine exposure potently increased oxygen consumption, whereas glutamine deprivation selectively decreased ATP concentration in the Rb TKO MEFs but not the wild-type (WT) MEFs. In addition, TKO MEFs exhibited elevated production of glutathione from exogenous glutamine and had increased expression of gamma-glutamylcysteine ligase relative to WT MEFs. Importantly, this metabolic shift towards glutamine utilization was required for the proliferation of Rb TKO MEFs but not for the proliferation of the WT MEFs. Last, addition of the TCA cycle intermediate α-ketoglutarate to the Rb TKO MEFs reversed the inhibitory effects of glutamine deprivation on ATP, GSH levels and viability. Taken together, these studies demonstrate that the Rb/E2F cascade directly regulates a major energetic and anabolic pathway that is required for neoplastic growth.