The purpose of this study is to investigate the change of phosphorylated p44/42 extracellular signal-regulated kinase (pERK1/2) and c-fos expression induced by single-prolonged stress (SPS) in medial prefrontal cortex (mPFC), and to determine whether extracellular signal-regulated kinase (ERK) pathway plays a role in SPS. Before exposure to SPS, Wistar rats were bilaterally infused with PD98059, inhibitor of ERK, into the mPFC. Animals were then tested in the open field (OF), elevated plus-maze (EPM), and Morris water maze (MWM). Brains were removed for immunohistochemical staining and Western blotting of pERK1/2. And reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect the c-fos mRNA, a member of immediate early genes (IGEs) family. SPS exposure resulted in pronounced anxiety-like behavior compared to control animals. SPS-exposed animals also displayed marked learning and spatial memory impairments. PD98059 significantly ameliorated anxiety-like behavior, learning, and spatial memory impairments. Expression of pERK1/2 in mPFC was significantly increased after rats were exposed to SPS, and the increase induced by SPS was significantly abolished by PD98059. The results of RT-PCR showed that the expression of c-fos mRNA had a significant increase in SPS rats compared with control rats, and the increase was significantly abolished by PD98059. The results suggest that pERK1/2 may be related to signal transduction pathway in single-prolonged stress.