To investigate the changes of meibomian gland (MG) and tear film stability in subjects with eyelid tattoos.Forty female subjects were recruited. Ten subjects had eyelid tattoos and 30 subjects did not (control group). Tear film break-up time (TBUT) measurement and fluorescein staining were performed to evaluate the tear film. Distances between the eyelid tattoo and the MGs were measured and used to assign a tattoo score. The overall tattoo score, defined as the sum of upper and lower lid tattoo scores, was determined for each eye. MG loss was scored for each eyelid and added together to obtain the total "meiboscore" for each eye using meibography. Values between the tattoo and control groups were examined and compared.The TBUT in the tattoo group (4.3 ± 0.9 seconds) was shorter than that in the control group (11.0 ± 4.3 seconds; P < 0.001). Corneal erosion, measured with fluorescein staining, was more severe in the tattoo group (1.6 ± 0.5) than that in the control group (0.2 ± 0.2; P < 0.001). MG loss was also more severe in the tattoo group (3.4 ± 1.5) than that in the control group (0.9 ± 0.6; P < 0.001). The total tattoo score was correlated with the total meiboscore (r = 0.852, P < 0.001, Spearman correlation coefficient).Eyelid tattooing shortened TBUT, increased fluorescein staining, and induced MG loss. Therefore, eyelid tattooing increases tear film instability through MG disturbance, and patients could have exacerbated signs and symptoms of ocular surface disease.