The long-term use of visual display terminals (VDT) is linked to an increased risk of dry eye disease (DED). Numerous studies have indicated that ocular mucins play a vital role in the pathogenesis of DED. Therefore, we aimed to evaluate (1) whether mRNA levels of membrane-associated mucins (MAMs), including MUC1, MUC4, MUC16, and MUC20, as well as MUC5AC are altered in conjunctival cells of VDT users with and without DED and (2) the relationship between mucin levels and subjective and objective tests of DED in VDT users.Seventy-nine VDT users were enrolled and divided into DED (n = 53) and control (n = 26) groups. All participants were evaluated for parameters of DED using the Ocular Surface Disease Index (OSDI) questionnaire, tear breakup time (TBUT), corneal fluorescein staining (CFS), lissamine green (LG) staining, and tear meniscus height (TMH). Based on the conjunctival impression cytology (CIC) method, differences in MUC1, MUC4, MUC16, MUC20, and MUC5AC mRNA expression levels were observed between the DED and control groups, and between symptomatic and asymptomatic participants.The DED group showed significantly decreased MUC1, MUC16, and MUC20 expressions (all P < 0.05) compared to the control group. In addition, these mucin levels were lower in subjects with frequent ocular symptoms (foreign body sensation, blurred vision and painful or sore eyes) than in asymptomatic participants (all P < 0.05). Correlation analysis revealed that MUC1, MUC16, and MUC20 levels in VDT users were positively correlated with TBUT or TMH, or both. However, no significant relationship was found between MUC4 and MUC5AC levels and the DED parameters.VDT users with an increased frequency of ocular discomfort or a diagnosis of DED had a decreased MUC1, MUC16 and MUC20 mRNA expression in their conjunctival cells. MAMs deficiency in the conjunctival epithelium may be one of the mechanisms leading to tear film instability and DED in VDT users.© 2023. The Author(s).