Substance P/high-affinity neurokinin-1 receptor (SP/NK1R) system plays a crucial role in the pathogenesis of dry eye disease (DED). NK1R antagonist can improve DED, but the mechanism of NK1R antagonist treating DED remains unclear. We examined the role of NK1R antagonist, CP-99,994 in DED model by possessing the phenol red cotton thread test, corneal fluorescein staining, and hematoxylin and eosin staining. Enzyme linked immunosorbent assay was performed to determine the concentration of inflammatory factors. Additionally, RNA sequencing, enrichment analysis and protein-protein interaction network were employed to identify the key targets. Real-time quantitative PCR and western blot analysis were utilized to determine the expression of hub genes. Plk1 inhibitor, GSK461364 was applied to explore the treatment mechanism of CP-99,994. The NK1R antagonist CP-99,994 alleviated dry eye symptoms and the concentrations of IL-6, IL-1β, and TNF-α were significantly decreased after CP-99,994 treatment. We obtained 68 differentially expressed genes after CP-99,994 treatment by RNA sequencing and pyroptosis-related genes (Plk1, Cdc25c, Cdk1) were identified from protein-protein interaction network as key targets of CP-99,994 treating DED. The expression levels of the Plk1, Cdc25c, and Cdk1 were significantly upregulated in the DED group, and CP-99,994 downregulated the expression of Plk1, Cdc25c, and Cdk1. Moreover, Plk1 inhibitor considerably promoted the therapeutic effect of CP-99,994 on DED model by reducing the release of IL-6, IL-1β, and TNF-α. The NK1R antagonist, CP-99,994 mitigated DED symptoms via inhibiting Plk1-Cdc25c-Cdk1 axis, which served as a novel therapeutic target for DED treatment.