Several lines of evidence indicate that phosphatidylinositol 3-kinase (PI3K) and PI3K-protein kinase B/Akt (PKB/Akt) signal pathway mediate the pain hypersensitivity induced by intradermal injection of capsaicin or nerve growth factor. However, the role of PI3K and PI3K-PKB/Akt signal pathway activation in neuropathic pain is still unclear. Using L5 spinal nerve ligation (L5 SNL) and immunohistochemistry, we found that the numbers of phospho-PKB/Akt-immunoreactive (p-PKB/Akt IR) positive neurons were significantly increased in ipsilateral L5 dorsal root ganglia (DRG) and adjacent L4 DRG started at 12 h after surgery and maintained to the 3rd day. Meanwhile, L5 SNL also induced an increased expression of p-PKB/Akt in ipsilateral L5 spinal dorsal horn. Double immunofluorescence staining showed that p-PKB/Akt expressed entirely in DRG neurons, especially in IB4-positive neurons. Intrathecal injection of PI3K inhibitor wortmannin or LY294002 and PKB/Akt inhibitor Akt inhibitor IV or (−)-Deguelin, started before L5 SNL, reduced the behavioral signs of neuropathic pain. Intraperitoneal injection of wortmannin or (−)-Deguelin as above also reduced the pain hypersensitivity. Post-treatment with wortmannin, started at the 1st day or the 3rd day after L5 SNL, decreased abnormal pain behaviors. Whereas the inhibitory effect of Akt inhibitor IV on established neuropathic pain was observed only in those rats that received the drug treatment started at the 1st day. Immunohistochemistry revealed that intrathecal injection of wortmannin significantly inhibited the activation of PKB/Akt in L5 DRG and L5 spinal cord. The data suggested that PI3K and PI3K-PKB/Akt signal pathway activation might contribute to the development of neuropathic pain.