Background: Ischemic stroke (IS) occurs when a vessel supplying blood to the brain is obstructed. Our previous study showed that D-allose exerts neuroprotective effects against acute cerebral ischemic/reperfusion (I/R) injury by reducing neuroinflammation. Here, we aim to clarify the beneficial effects and underlying mechanism of D-allose in suppressing IS-induced neuroinflammation damage, cytotoxicity and neuro-specific apoptosis and neurological deficits in vitro and in vivo. Methods: For in vivo study, the I/R model was induced by middle cerebral artery occlusion and reperfusion (MCAO/R) in C57BL/6J mice and D-allose was given intraperitoneal injection within 5min after reperfusion. In vitro, mouse hippocampal neuronal cells (HT22) with oxygen–glucose deprivation and reperfusion (OGD/R) were established as the cell model of IS. The neurological score evaluation, some cytokines, cytotoxicity and apoptosis in the brain and cell lines were measured. Moreover, Gal-3 short hairpin RNA, lentivirus and adeno-associated virus were employed to modulate Gal-3 expression in neuronal both in vitro and in vivo to reveal the molecular mechanism. Results: D-allose alleviated cytotoxicity, including cell viability, LDH release and apoptosis in HT22 cells after OGD/R, which also relieved brain injury, such as lesion volume, brain edema, neuronal apoptosis, as well as neurological functional deficits in a mouse model of I/R. Importantly, loss of Gal-3 enhanced the D-allose’s protection against I/R-induced HT22 cell injury, inflammation insults and apoptosis, whereas activation of TLR4 by selective agonist LPS increased the degree of injury of neuron, as well as abolished the protective effects of D-allose.