Polydatin (PD, also named pieceid, (E)‐piceid, (E)‐polydatin, trans‐polydatin, 3,4′, 5‐trihydroxystilbene‐3‐b‐D‐glucoside) is a monocrystalline compound originally isolated from the root and rhizome of Polygonum cuspidatum Sieb. et Zucc. (Polygonaceae), a traditional East Asia medicine. Previous study demonstrated that Polydatin has antioxidant and anti‐inflammatory effect. However, the effect of Polydatin in dry eye diseases (DED) has not been mentioned. We investigated the effect of Polydatin in the models of dry eye in vivo and in vitro. The measurement of tear volume showed that the decreased tear volume in the extraorbital lacrimal gland excised rats was significantly recovered by the Polydatin 0.5% eye drops. In addition, the corneal irregularity and lissamine green staining score revealed that Polydatin eye drops (0.05% and 0.5%) remarkably inhibited the increased corneal irregularity and lissamine green staining scores by the extraorbital lacrimal gland. Tear film breakup time assay showed that the shortened tear film breakup time was reasonably increased in the Polydatin 0.5% eye dropped group. Moreover, we examined the effect of Polydatin in the hyperosmolarity‐induced human conjunctival epithelium. The results from realtime PCR demonstrated that Polydatin significantly inhibited the mRNA expression of TNF‐α, IL‐6 and MMP9 in a concentration dependent manner. Taken together, our results suggest that Polydatin may a potential therapeutic agent to treat dry eye disease through the recovery of damaged corneal layer and inhibition of inflammation in conjunctival epithelium.