Diabetic neuropathic pain, an important micro vascular complication in diabetes mellitus, has been recognized as one of the most difficult types of pain to treat. Lack of understanding of etiology involved, inadequate relief, development of tolerance and potential toxicity of classical anti-nociceptive agents warrants the investigation of newer agents to relieve this pain. The aim of the present study was to explore the anti-nociceptive effect and possible mechanism of action of a serotonin reuptake inhibitor, fluoxetine, in streptozotocin (STZ)-induced diabetic mice. Diabetes was induced in mice by intraperitoneal injection of streptozotocin (STZ, 200 mg/kg). After 4 weeks of a single intraperitoneal injection of STZ, diabetic mice were subjected to tail-immersion and hot plate assay. Diabetic mice exhibited a significant hyperalgesia as compared to control mice. When diabetic mice were injected with fluoxetine (10 and 20, but not 5 mg/kg, i.p.) produced anti-nociceptive effect in both tail immersion and hot plate assays. The %MPE produced by fluoxetine (20 mg/kg/i.p) was significantly less in diabetic mice as compared with that in control mice. The antinociceptive effect of fluoxetine (20 mg/kg) in diabetic mice was dose dependently potentiated by pindolol (5 and 10 mg/kg, i.p., a selective 5-HT1A/1B receptor antagonist), attenuated by ritanserin (1 and 2 mg/kg, i.p., a selective 5-HT2A/2C receptor antagonist) and remained unaffected by ondansetron (1 and 2 mg/kg, i.p., a selective 5-HT3 receptor antagonist) in both test systems. These results suggest that fluoxetine-induced antinociception primarily involves serotonin pathway modulation through 5-HT1 and 5-HT2 receptors, but not through 5-HT3 receptors in chronic pain associated with STZ-induced diabetic neuropathic pain. Furthermore, the potentiation of anti-nociceptive effect of fluoxetine by pindolol indicates the usefulness of combination of an antidepressant and a 5-HT1A/1B receptor antagonist in therapy of human diabetic neuropathic pain.