Objective To investigate the effect of co-culture of microglial cells (MGs) and neural stem cells (NSCs) with overexpression of nuclear receptor-related factor 1 (Nurr1) gene on the differentiation of NSCs into dopaminergic neurons.Methods Primary NSCs and MGs were cultured, and the overexpression of Nurr1 gene was performed. Cell Counting Kit-8 (CCK-8) assay was used to investigate the influence of Nurr1 overexpression on the viability of NSCs and MGs. The Transwell co-culture system was used for the co-culture of NSCs and MGs, and the cells were divided into NSC group, NSC+MG group, and N(NSC+MG) group. ELISA was used to measure the expression of brain-derived neurotrophic factor (BDNF), platelet-derived neurotrophic factor (PDNF), and glial cell-derived neurotrophic factor (GDNF) on days 3, 6, and 9 of co-culture; RT-PCR and Western blot were used to measure the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT), and Nurr1 on day 9; cell immunofluorescence was used to determine cell differentiation and the numbers of TH⁺ and DAT⁺ cells, and the efficiency of the differentiation of NSCs into dopaminergic neurons was calculated.Results Primary NSCs and MGs were successfully cultured, and the overexpression of Nurr1 gene was successfully performed. The CCK-8 assay showed that Nurr1 overexpression had no significant influence in the viability of NSCs and MGs. The results of ELISA showed that the N(NSC+MG) group had significantly higher expression of BDNF, PDNF, and GDNF at different time points than the other two groups (P<0.05). RT-PCR and Western blot showed that the N(NSC+MG) group had significantly higher expression of TH, DAT, and Nurr1 than the other two groups (P<0.05). Cell immunofluorescence showed that the N(NSC+MG) group had a significantly higher percentage of TH+ cells than the other two groups (P<0.05).Conclusions Nurr1 can promote the secretion of neurotrophic factors in the co-culture system of NSCs and MGs, and overexpression of Nurr1 can promote the differentiation of NSCs into dopaminergic neurons in this co-culture system.