Activation of extracellular signal-regulated kinase (ERK), a mitogen activated-protein kinase (MAPK), in dorsal horn neurons contributes to inflammatory pain by transcription-dependent and -independent means. We have now investigated if ERK is activated in the spinal cord after a spinal nerve ligation (SNL) and if this contributes to the neuropathic pain-like behavior generated in this model. An L5 SNL induces an immediate (<10 min) but transient (<6 h) induction of phosphoERK (pERK) restricted to neurons in the superficial dorsal horn. This is followed by a widespread induction of pERK in spinal microglia that peaks between 1 and 3 days post-surgery. On Day 10, pERK is expressed both in astrocytes and microglia, but by Day 21 predominantly in astrocytes in the dorsal horn. In the L5 DRG SNL transiently induces pERK in neurons at 10 min, and in satellite cells on Day 10 and 21. Intrathecal injection of the MEK (ERK kinase) inhibitor PD98059 on Day 2, 10 or 21 reduces SNL-induced mechanical allodynia. Our results suggest that ERK activation in the dorsal horn, as well as in the DRG, mediates pain through different mechanisms operating in different cells at different times. The sequential activation of ERK in dorsal horn microglia and then in astrocytes might reflect distinct roles for these two subtypes of glia in the temporal evolution of neuropathic pain.