Neurokinin peptides neurokinin‐1 (NK1), neurokinin‐3 (NK3), and related receptors are abundantly distributed in the substantia nigra (SN) and evidenced by their possible roles in the Parkinson’s disease. Differential intervention roles of NK3 on kainic acid (KA)‐induced neuronal injury in the SN of mice were thus in vitro and in vivo studied by Fluoro‐Jade C (FJC) staining, immunohistochemistry to tyrosine hydroxylase (TH) or phospho‐NMDA receptor, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. It revealed that (i) in contrast to protective effect of NK1 agonist septide that reduced FJC‐positive degenerative neurons and lesion volume insulted by KA, NK3 agonist senktide significantly increased FJC‐positive ones and lesion volume, and this effect was sufficiently reversed by NK3 antagonist SB218795; (ii) similarly, senktide reduced TH‐positive neurons and this effect was antagonized by SB218795, but septide increased TH‐positive ones; (iii) mechanistic observation showed differential influences of NK1 and NK3 agonists on phosphorylated‐NMDA receptor subunit 1 (phospho‐NMDAR1) and glial fibrillary acidic protein‐expressing astrocytes, i.e. senktide enhanced of NMDA receptor phosphorylation and astrocyte activity, while septide reduced NMDA receptor phosphorylation and astrocytic response; (iv) cell culture further confirmed the exacerbating effect of NK3 agonist on KA‐induced lesion of nigral cells or dopaminergic neurons, in which administration of senktide alone did not show significant cell toxicity. This study presents new evidence that neurokinin NK3 instead of NK1 synergistically exacerbate excitotoxic neuronal degeneration in the SN in a dose‐dependent manner and possibly through modulation of NMDA receptor phosphorylation and astrocyte activity, suggesting their potential significance in novel pharmaceutical therapy against Parkinson’s disease.