By knocking down E4orf1 gene expression in Ad36-infected cells, Experiment 1 determined that Ad36 ��requires�� its E4orf1 protein for up-regulating cellular glucose uptake. Next, by inducibly expressing only E4orf1 in cells, Experiment 2 identified E4orf1 as ��sufficient�� to up-regulate the Ras pathway and glucose uptake. Experiment 3 revealed that similar to the action of E4orf1 of Ad9, Ad36 E4orf1 may activate Ras by binding to Dlg1 protein. Moreover, total Ras and particularly, the H-Ras isoform is significantly increased and activated by Ad36 E4orf1. By mutating the PBM of Ad36 E4orf1, Experiment 4 showed that E4orf1 requires its PBM to activate Ras or to increase glucose uptake. Finally, Experiment 5 determined that transient transfection by E4orf1 significantly increases glucose uptake in preadipocytes, adipocytes, and myoblasts, and significantly reduces glucose output by hepatocytes. Ad36 infection improves glycemic control in chow-fed Evofosfamide normoglycemic rats and mice and in high fat fed hyperglycemic mice. Natural infection with Ad36 predicts better glycemic control in normoglyemic and diabetic humans. The virus appears to exert its anti-hyperglyemic action by increasing glucose uptake by preadipocytes, adipocytes, and myocytes, and by reducing hepatic glucose output. Ras/PI3K pathway activation is required for Ad36-induced cellular glucose uptake. These findings are potentially highly significant for developing new treatment approaches for type 2 diabetes and insulin resistance. Particularly, the unique capability of Ad36 to attenuate hyperglycemia despite a continued HF-diet and without a reduction in visceral or subcutaneous adiposity offers a remarkable opportunity to creatively negate the hyperglycemic effects of excess adiposity or dietary fat intake, without the need to reduce it. However, for developing a therapeutic approach, infection with a virus is impractical. Instead, a viral protein that is responsible for the effect could provide a drug ligand or a target. Here we show that E4orf1 is required to mediate the glucose uptake induced by Ad36. Also, E4orf1 is sufficient to promote glucose uptake in preadipocytes, adipocytes, and myoblasts, and to reduce glucose output by hepatocytes. Ad9 E4orf1, which is 96% homologous to Ad36 E4orf1, mediates Ras activation by complexing with Dlg1 via its PBM , which also appears to be the case with Ad36 E4orf1. Ad36 E4orf1 activates Ras and PI3K, the two main signaling components required for Ad36 AbMole BioScience infection-induced glucose disposal. Ad36 E4orf1 requires its PBM for activating Ras and for upregulating glucose uptake. Specifically, Ad36 E4orf1 increases the relative abundance and activation of H-Ras isoform.