Purpose: To compare the degree of tear film instability and severity of meibomian gland dysfunction between subjects who use eyeliner and those who do not use eyeliner., Methods: This cross-sectional study included 42 healthy volunteer women who had no dry eye symptoms (Ocular Surface Disease Index score < 13) and aged between 18 and 40 years. The subjects were classified into 2 groups: an eyeliner-use group (EL: regularly used eyeliner ≥3 d/wk and continuously used ≥6 mo) and a noneyeliner-use group as controls. A questionnaire for ocular surface symptoms using a visual analog scale was administered. Then, a number of eye tests were performed [grading of conjunctival inflammation, fluorescein tear breakup time, ocular surface fluorescein staining, Schirmer I, evaluation of meibomian gland (MG) function, detection of eyelid margin abnormalities, and Demodex detection]., Results: Tear breakup time was significantly lower in the EL group compared with controls (3.0 ± 1.9 vs. 5.8 ± 2.1 s, P < 0.001). MG grading was significantly higher in the EL group than in controls (P = 0.004); higher grade (grades 2-3) was found in 85.7% of EL and 47.6% of controls. Meiboscore was also higher in EL than in controls (P = 0.001). Regarding the morphological changes in lid margin, only telangiectasia was detected significantly more in EL (28.6%) compared with controls (4.8%) (P = 0.041). Conjunctival inflammation was observed 4 times more in EL (66.7%) than in controls (14.3%), P = 0.001. Other outcomes included ocular surface symptoms and fluorescein staining scores, and Schirmer I and Demodex detection were not significantly different between both groups., Conclusions: The regular use of eyeliner induces tear film instability and MG dysfunction.