Hyperosmolarity is pro-inflammatory stress to the ocular surface epithelium for dry eye disease (DED). Astaxanthin (AST) is a kind of carotene, which exists in seafood and has been proved to inhibit inflammation of numerous diseases. The aim of this study was to characterize the protective effect and potential mechanism of AST on DED in vitro and in vivo. The mice models and human corneal epithelial cells (HCECs) cultures exposed to hyperosmotic saline solution (HOSS) in vitro and in vivo experiments, respectively. Experimental subjects first pretreated with AST, and then the effect of the compound was assessed with clinical evaluation, real-time PCR (RT-PCR), Western blot and immunofluorescent staining. We further investigated the possible mechanism of AST in DED by pre-treating with phosphoinositide 3-kinase inhibitor (LY294002). The addition of AST significantly reduced the expression of High-mobility group box 1 (HMGB1), as well as significantly inhibited the increases of TNF-α, IL-1β in a dose-dependent manner, but could promoted the expression of phospho-Akt (p-Akt). BALB/c mice in DE group pretreated with AST showed significantly decreased corneal fluorescein staining scores. Moreover, Pretreatment with LY294002 could eliminate the effects of AST preconditioning on the decrease of HMGB1. Our study provides evidence that AST could ameliorate DED which may be related to the inhibition of HMGB1, TNF-α, IL-1β, while PI3K/Akt signaling pathway may be involved in the expression of HMGB1 and the protective effect of AST preconditioning.Copyright © 2020. Published by Elsevier Ltd.