Abstract: Objective To investigate the activity of protein kinase B (Akt) and its downstream protein, glycogen synthase kinase-3β (GSK-3β) under hypoxia-ischemia (HI), and the possible regulation for axonal density. Methods Postnatal day 10 SD rats were suffered the right common carotid artery ligation and 8% mixture of oxygen and nitrogen hypoxia 2.5 h to produce HI model. The expression of total and phosphorylated Akt and GSK-3β was detected by western blot after HI. After pretreatment of Akt inhibitor, wortmannin or LY294002, Western blot detect the expression of total and phosphorylated of Akt, GSK-3β at 4 h and 24 h after HI. After pretreatment of wortmannin, axonal density was determined by Bielschowsky silver impregnation, and histological injury was evaluated by hematoxylin and eosin (HE) staining. Results The expression of total Akt and GSK-3β remained unchanged after HI. p-Akt protein significantly decreased at 0.5 h, increased at 2 h and reached the highest at 4 h, returned to baseline at 8 h, declined at 24 and 48 h after HI, and finally returned to baseline again at 72 h compared with that of sham controls. p-GSK-3β protein decreased at 0.5 h, increased at 2 h, reached the highest at 4 h, returned to baseline at 8 and decreased at 24 h, reached the lowest at 48 h, and returned to baseline at 72 h. Wortmannin or LY294002 intervention didn't change the expression of total Akt and GSK-3β, while decrease the p-Akt and p-GSK-3β expression. HI cause decreased axonal density, and the histological injury of brain. Wortmannin pretreatment could aggravate the histological injury and decrease axonal density after HI. Conclusion The Akt pathway is involved in axonal density and histological brain injury after HI in neonatal rat.