Calorie restriction (CR) prevents obesity and exerts anticancer effects in many preclinical models. CR is also increasingly being used in cancer patients as a sensitizing strategy prior to chemotherapy regimens. While the beneficial effects of CR are widely accepted, the mechanisms through which CR affects tumor growth are incompletely understood. In many cell types, CR and other nutrient stressors can induce autophagy, which provides energy and metabolic substrates critical for cancer cell survival. We hypothesized that limiting extracellular and intracellular substrate availability by combining CR with autophagy inhibition would reduce tumor growth more effectively than either treatment alone.A 30 % CR diet, relative to control diet, in nude mice resulted in significant decreases in body fat, blood glucose, and serum insulin, insulin-like growth factor-1, and leptin levels concurrent with increased adiponectin levels. In a xenograft model in nude mice involving H-Ras(G12V)-transformed immortal baby mouse kidney epithelial cells with (Atg5 (+/+) ) and without (Atg5 (-/-)) autophagic capacity, the CR diet (relative to control diet) genetically induced autophagy inhibition and their combination, each reduced tumor development and growth. Final tumor volume was greatest for Atg5 (+/+) tumors in control-fed mice, intermediate for Atg5 (+/+) tumors in CR-fed mice and Atg5 (-/-) tumors in control-fed mice, and lowest for Atg5 (-/-) tumors in CR mice. In Atg5 (+/+) tumors, autophagic flux was increased in CR-fed relative to control-fed mice, suggesting that the prosurvival effects of autophagy induction may mitigate the tumor suppressive effects of CR. Metabolomic analyses of CR-fed, relative to control-fed, nude mice showed significant decreases in circulating glucose and amino acids and significant increases in ketones, indicating CR induced negative energy balance. Combining glucose deprivation with autophagy deficiency in Atg5 (-/-) cells resulted in significantly reduced in vitro colony formation relative to glucose deprivation or autophagy deficiency alone.Combined restriction of extracellular (via CR in vivo or glucose deprivation in vitro) and intracellular (via autophagy inhibition) sources of energy and nutrients suppresses Ras-driven tumor growth more effectively than either CR or autophagy deficiency alone. Interventions targeting both systemic energy balance and tumor-cell intrinsic autophagy may represent a novel and effective anticancer strategy.