Background: Hyperglycemia triggered endoplasmic reticulum (ER) stress is one of the major causes for platelet hyperactivation and apoptosis in diabetes mellitus (DM). Reticulon-4B (aka Nogo-B) mainly localizes to the ER, and has been shown to influence the ER morphology, ER-Golgi trafficking, apoptotic balance, vesicle formation and protein trafficking in cells. The present study is aimed to investigate the role of Nogo-B on platelet function in DM. Methods and Results: Nogo-B is highly expressed in platelets from healthy individual. Platelets from DM patients and diabetic mice have decreased Nogo-B level. Using Streptozotocin (STZ) induced diabetic mouse model, we show that loss of Nogo (Nogo-/- mice) decreased platelet number, increased mean platelet volume and prolonged bleeding time compared to wild-type (WT) mice. Platelets from Nogo-/- mice were hyperactive with higher JONA and P-selectin surface expression compared to WT mice. Loss of Nogo increased thrombin and collagen induced platelet aggregation. Furthermore, platelets from diabetic Nogo-/- mice show elevated reactive oxygen species (ROS) production, decreased mitochondria membrane potential and increased apoptosis, which can be rescued by antioxidant N-acetyl-L-cysteine. Mechanistically, we show Nogo-B prevented sequestration of antiapoptotic proteins Bcl-xL and Bcl-2 induced by hyperglycemia, subsequently protected against platelet mitochondrial damage, ROS production, caspase-3 activation and apoptosis. Conclusion: These findings demonstrate that Nogo-B protects against ER stress induced platelet apoptosis and hyperactivation in DM by regulating Bcl-xL and Bcl-2 sequestration and mitochondrial damage. This novel pathway may provide therapeutic targets for thrombotic complications in diabetes mellitus.