Human activities in the areas of high altitude have increased significantly recently. Brain is highly sensitive to changing of oxygen pressure due to high altitude, and this physiological response may lead to serious brain injury, such as learning and memory disabilities. Puerarin is a phytoestrogen with many pharmacological activities, such as treatment of neurological disorders. However, most of current drugs can not easily enter brain through the blood-brain barrier (BBB). The nose-to-brain route can bypass BBB for brain-targeting. Here, thermosensitive in situ hydrogels (TISGs) of puerarin were prepared with poloxamers 407, poloxamers 188 and propylene glycol to improve bioavailability and brain targeting. In vitro drug release in simulated nasal fluids, rheological properties and cilia toxicity of puerarin TISGs were explored. The pharmacodynamics and pharmacokinetics of puerarin by intranasal (i.n.) and oral (p.o.) administrations were also evaluated. The viscosity of puerarin TISGs tended to increase obviously with increased temperature. The puerarin release profile and transmucosal process of puerarin TISGs could be described with the first-order kinetics equation, depending on drug diffusion. The cilia toxicity of puerarin TISGs was not obvious. Rat models of hypobarism/hypoxia-induced brain injury were established with a hypobaric simulation chamber. Morris water maze and open filed tests indicated that puerarin TISGs improved the spatial memory and spontaneous exploratory behavior of the rats suffering from hypoxia-induced brain injury. Furthermore, puerarin TISGs decreased the level of oxidative stress cytokines (malondialdehyde (MDA) and glutathione (GSH)) in the peripheral circulation, alleviated the cerebral histological lesions, and relieved the expression of hypoxia-inducible factor-1α (HIF-1α). Intranasal puerarin TISGs were absorbed quickly with a shorter T (10.0 ± 5.7 min) compared to that of oral puerarin (36 ± 13.4 min). In addition, the relative bioavailability of i.n. puerarin TISGs was high to 300% compared to oral administration of puerarin. The area under the curve (AUC) of brain after i.n. administration of puerarin TISGs was 954.5 ± 335.1 h.ng/mL, while no puerarin was detected in the brain after oral administration. Therefore, i.n. puerarin TISGs led to excellent brain targeting effect. Puerarin TISGs are an effective neuroprotector formulation for prevention of brain injury induced by acute high-altitude hypoxia.Copyright © 2020. Published by Elsevier B.V.