Sublethal preconditioning ischemia protects neurons from lethal ischemia, and activation of ERK is associated with this protection. However, sublethal ischemia and reperfusion also results in rapid inactivation of ERK, which contributes to the dual-phase activation profile of ERK. In the present study, we observed sublethal ischemia-induced rapid inactivation of ERK was accompanied by phosphorylation of Raf-1 at Ser289/296/301 sites. Inhibition of calcium signaling by ketamine resulted in down-regulation of the Raf-1/ERK cascade and decreased phosphorylation of Raf-1 at Ser289/296/301. The MEK inhibitor U0126 suppressed ERK activity and phosphorylation of Raf-1 at Ser289/296/301 but not Raf-1 activation elicited by its dephosphorylation at S259 following ischemia. The PP2A inhibitor cantharidin but not Pin1 inhibitor juglone blocked Raf-1 dephosphorylation at Ser289/296/301 and ERK dephosphorylation and led to ERK sustained activation, which is associated with transcriptional up-regulation of genes related to differentiation. Furthermore, dual-phase activation of ERK did not alter the mRNA levels of genes related to proliferation or differentiation. These results indicate the initial robust activation of ERK phosphorylates Raf-1 at Ser289/296/301, resulting in Raf-1inhibition and then prompt inactivation of ERK following sublethal preconditioning ischemia. Dual-phase activation of ERK may exert its neuroprotection against lethal ischemia through blocking cell proliferation and differentiation.