Post-stroke depression (PSD) is one of the most familiar complications of stroke, which refers to stroke patients who have varying degrees of depression (lasts for >2 weeks). SET domain-containing 3 (SETD3) is a conserved histone H3 methyltransferase, and the role of SETD3 in some diseases is increasingly being explored. However, the effects of SETD3 in PSD remain unclear. In this study, the PSD rat model was firstly constructed by Endothelin-1 injection combined with chronic unpredictable mild stress, and we discovered that SETD3 expression was up-regulated in PSD rat model. Additionally, SETD3 knockdown relieved the depressive symptom of PSD. Moreover, SETD3 knockdown promoted proliferation and differentiation of neural stem cells (NSCs). Due to the critical role of vascular endothelial growth factor (VEGF) in antidepressant and SETD3 can negatively regulate VEGF, we speculated that SETD3 may regulate PSD progression through VEGF. Our results demonstrated that SETD3 knockdown up-regulated VEGF expression. Furthermore, SETD3 modulated the proliferation and differentiation of NSCs through regulating VEGF expression. In conclusion, our study indicated that up-regulation of SETD3 contributed to PSD progression in rats through negatively regulating VEGF expression. The findings of this work suggest that SETD3 may be a promising target for treating PSD in the future.