Polymorphisms located within NOS3 gene have been investigated as susceptibility variants for diabetic nephropathy (DN) in type 2 diabetes mellitus (T2DM) in a large number of studies. However, these previous articles yielded inconsistent results and we aimed at elucidating the impact of NOS3 variants on DN risk in T2DM by conducting an updated systematic data synthesis. A total of 36 studies (12,807 participants) were selected for qualitative data synthesis, while 33 records with 11,649 subjects were included in the meta-analysis. The pooled analysis demonstrated the association of minor alleles of rs2070744 and rs1799983 with an increased susceptibility to DN (P < 0.001 and P = 0.015 for allelic model, respectively). For both of these variants, a significant effect of subgrouping according to ethnicity was found. Rs869109213 displayed an association with DN susceptibility, with pooled effect measures indicating a predisposing effect of the minor allele a (Prec = 0.002, ORrec = 1.960, 95%CI 1.288–2.983; Paavs. bb = 0.001, ORaavs. bb = 2.014, 95%CI 1.316–3.083). These findings support the effects of NOS3 variants on the risk of developing DN in T2DM. Display omitted •A total of 36 studies were selected for qualitative data synthesis.•33 records with 11,649 subjects were included in the meta-analysis.•Minor alleles of rs2070744 and rs1799983 associate with diabetic nephropathy risk.•Allele a of rs869109213 increases susceptibility to diabetic nephropathy.