PURPOSE: Inflammation and apoptosis contribute to the development of dry eye disease (DED) and meibomian gland dysfunction (MGD). This study aimed to investigate the effect of caffeine on the ocular surface and tear inflammatory cytokines through clinical, in vivo, and in vitro experiments. METHODS: In the clinical study, comprehensive ophthalmic examinations of participants in the control and the caffeine groups were compared, including ocular surface and tears inflammatory cytokines. For in vitro study, rat meibomian gland epithelial cells (RMGECs) and human corneal epithelial cells (HCECs) were pretreated with or without caffeine and then stimulated with lipopolysaccharide (LPS). Inflammatory responses, apoptosis, and differentiation in cells were analyzed. In vivo study, apolipoprotein E knockout (ApoE-/-) mice were given caffeine-diet or no caffeine-diet, and their meibomian glands (MGs) and corneal tissue were compared. RESULTS: Participants in the caffeine group exhibited significantly healthier ocular surface, lower tears inflammatory cytokines and a reduced prevalence of DED compared to the control group. In vitro study, caffeine pretreatment attenuated inflammatory responses, apoptosis and differentiation in LPS-induced RMGECs. Meanwhile, caffeine also markedly suppressed inflammatory responses and apoptosis in LPS-induced HCECs. In vivo study showed that ApoE-/- mice with caffeine-diet had more normal morphology of MGs and corneas compared to those without caffeine-diet, along with reduced inflammatory responses, cells apoptosis and ductal keratinization. Both in vitro and in vivo studies indicated that caffeine treatment was observed to inactivate of nuclear factor kappa B (NF-κB) phosphorylation. CONCLUSIONS: Our study indicated that caffeine may be a protective potential of ocular surface, providing a new perspective on clinical treatment for DED and MGD.