Although C-C motif chemokine ligand 2 (CCL2) plays a critical role in the pathogenesis of neuropathic pain through neuron-microglia interactions, its pronociceptive function underlying inflammatory pain remains to be fully understood. The present study aimed to elucidate the potential role of CCL2 in pain sensitization following zymosan-induced inflammation. Intraplantar injection of zymosan into the rat hind paw significantly increased the expression of CCL2 in both dorsal root ganglions and the superficial dorsal horn. The expression of CCL2 was exclusively present in the isolectin B4-positive unmyelinated primary afferent fibers, but no in other cells of the spinal cord. Intrathecal administration of RS504393 (a CCR2 antagonist) markedly reduced the zymosan-induced thermal and mechanical hyperalgesia accompanied with reduced expression in the spinal cord of c-Fos, CD11b, phosphorylated p38 mitogen activated protein kinases (p-p38), and interleukin-1β. Whole cell patch-clamp recordings on spinal cord slices further revealed that the incubation of CCL2 evoked an evident inward current in substantia gelatinosa neurons and increased level of p-p38 in microglia. Moreover, co-incubation with minocycline (an inhibitor of microglial activation) prevented CCL2-mediated activation in the spinal cord slice. Taken together, we propose that the increased CCL2 expression from primary afferent fibers following zymosan-induced inflammation activates nociceptive neurons in the spinal dorsal horn via a microglia-dependent mechanism.