Painful Diabetic Neuropathy (PDN) is one of the most common and annoyingcomplications of diabetes mellitus. The pathogenesis of PDN is complex and still unclear.Recently it has become clear that nitric oxide (NO) and proinflammatory cytokines playan important role in the pathogenesis and maintenance of pain in PDN. Based on thisphenomenon, this study was conducted to investigate whether the cytokine tumornecrosis factor-alpha (TNF-α) and NO, in this case inducible Nitric Oxide Synthase(iNOS), play a role in PDN pathogenesis. The study was carried in two steps. The first step was a cross sectional and the second step was a case-control study. The study was performed in 110 type-2 diabetic patients. The plasma TNF-α levels were determined by ELISA while the expression of TNF-α and iNOS in mononuclear cells were analyzed immunohistochemically. Of 110 subjects, 59 patients suffered from Painful DN (case) and the remaining 51 patients were Painless DN (control). Cross sectionally, plasma TNF-α levels and immunoreactivity for iNOS and TNF-α were higher in patients with more severe pain in the Visual Analog Scale (VAS). There were statistically significant differences (p < 0.05) between mild and severe pain in regard to TNF-α level (15.24 pg/ml ± 5.42 vs. 20.44 pg/ml ± 10.34 ); to iNOS immunoreactivity (9.72 % ± 8.61 vs. 15.6% ± 11.84); and to TNF-α immunoreactivity (13.0 % ± 9. 48 vs. 20.44% ± 11.75). The case control study showed that TNF-α had an odd ratio of 5.053 [CI 95% (2.241-11.392); p < 0.001]. TNF-α immunoreactivity of 4.125 [CI 95% (1.805-9.425); p < 0.001]; and iNOS immunoreactivity of 3.546 [CI 95% (1.613-7.795); p = 0.002]. There were correlations between TNF-α level, TNF-α and iNOS immunoreactivity and VAS with coefficient correlation: 0.330; 0.285 and 0.275 (p < 0.05) respectively. It is concluded that Diabetic Neuropathy patients with high TNF-α levels, iNOS and TNF-α immunoreactivity of mononuclear cells have higher risk for painful DN than painless DN. The higher TNF-α level, iNOS and TNF-α immunoreactivity the more severe was the pain. This supports the hypothesis that TNF-α and iNOS have role in PDN pathogenesis. The results of this research could be applied as a basic for further research in pursuit of better management of PDN