Our previous work has shown that peri-sciatic administration of recombinant rat TNF-α (rrTNF) induces mechanical allodynia and up-regulation of TNF-α in the spinal dorsal horn of rats; however, the underlying mechanisms remain unknown. In the current study, we found that the levels of phosphorylated Src-family kinases (p-SFKs) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were significantly increased in bilateral lumbar spinal dorsal horn on day 3 after rrTNF administration. Double immunofluorescence staining revealed that p-SFKs and p-p38 MAPK were nearly restricted to the microglia. Intrathecal delivery of SFKs inhibitor PP2 or p38 MAPK inhibitor SB203580, started 30min before rrTNF administration and given once daily thereafter for 7 days, blocked mechanical allodynia in bilateral hind paws and increase of TNF-α expression in the spinal dorsal horn. Moreover, PP2 inhibited the up-regulation of p-p38 MAPK induced by rrTNF. We also found that intrathecal injection of TNF-α neutralization antibody alleviated mechanical allodynia in bilateral hind paws and suppressed up-regulation of p-SFKs and p-p38 MAPK. These results suggest that activation of the SFKs/p38 MAPK pathway in microglia and subsequent TNF-α expression in the spinal dorsal horn may contribute to the mechanical hyperalgesic state induced by peri-sciatic administered rrTNF.