Background Hyperosmosis stress (HS) was a key pathological factor in the development of dry eye disease (DED). Nicotinamide mononucleotide (NMN) demonstrated protective effects in the corneal damage, however, its role in the HS-induced DED remained unclear. Methods A NaCl based HS in-vitro model (500 mOsm) was generated and used in a co-culture system including corneal epithelial cells (CEC) and macrophage cell line RAW264.7. The effect of NMN on NAD+ metabolism and the expression of HS biomarker, tonicity-responsive element binding protein (TonEBP), was studied in the CEC. The cellular activity, including cell viability, apoptosis status and lactate dehydrogenase (LDH) release through trypan blue staining, flow cytometry and LDH assay, respectively. The mitochondrial membrane potential (MMP) assay would be conducted using the JC1 kit. The expression of IL-17a were detected using RT-PCR, ELISA and Western blot. After co-culture with the CEC in different group for 24 h, the phagocytosis ability and macrophage polarization were assessed in RAW264.7 cells co-cultured with CEC with or without HS or NMN treatment. Besides, the involvement of Notch pathway in the RAW264.7 would be analyzed. The potential involvement of Sirtuin 1 (SIRT1) and IL-17a in the crosstalk between CEC and macrophage was studied with SIRT1 inhibitor EX 527 and anti-IL-17a monoclonal antibody, respectively. Results NMN treatment increased NAD+ concentration and thus improved cell viability, reduced apoptotic rate and decreased the LDH release in HS-treated CEC. Besides, NMN alleviated HS-induced MMP, intracellular ROS and LDH release. Besides, it was confirmed NMN improve SIRT1 function and decreased the HS related IL-17a expression in CEC and then alleviated macrophage phagocytosis ability and M1 polarization based on a CEC-macrophage co-culture system. Moreover, NMN treatment of CEC in the CEC could moderate the subsequent macrophage activation through Notch pathway. SIRT1 activation and IL-17a inhibition was regarded as key progress in the function of NMN based on the application of EX 527 and anti-IL-17a antibody in the CEC-macrophage co-culture system. Conclusion The findings demonstrated that NMN could alleviated HS-induced DED status through regulating the CEC/macrophage interaction. Our data pointed to the role of SIRT1, IL-17a and Notch pathway in the function of NMN and then provided updated knowledge of potential NMN application in the management of DED.