A large body of evidence indicates that nitric oxide (NO) plays an important role in the processing of persistent inflammatory and neuropathic pain in the spinal cord. Several animal studies revealed that inhibition or knockout of NO synthesis ameliorates persistent pain. However, spinal delivery of NO donors caused dual pronociceptive and antinociceptive effects, pointing to multiple downstream signaling mechanisms of NO. This review summarizes the localization and function of NO-dependent signaling mechanisms in the spinal cord, taking account of the recent progress made in this field.