Objective: To explore the effect of androgen replacement therapy in a rabbit dry eye model characterized by androgen deficiency and meibomian gland dysfunction (MGD). Methods: Thirty 6-month-old male Chinchilla rabbits were randomly divided into the treatment group, model group and control group, with 10 rabbits in each group. In the treatment and model groups, 2/3 of the meibomian gland openings were closed by cauterization with electric coagulation pen, and bilateral testes were removed. One gram gel containing 1% testosterone was applied for 28 days on the skin of rabbits in the treatment group since day 28 after the surgery. The model group and control group received transdermal petrolatum instead. Tear secretion, tear breakup time (TBUT), corneal fluorescein staining, and serum free testosterone level were monitored throughout the study period. The globes and eyelids were collected for hematoxylin-eosin staining and periodic acid-Schiff staining. Conjunctival tissue was tested for the expression of miRNA-744-5p and interleukin-6. Meibum was collected for fatty acid analysis. Results: Animals presented with typical dry eye signs and androgen deficiency. After 28-day androgen replacement therapy, compared with the model group, the treatment group had a significantly higher tear secretion rate [(14.7±5.2) vs (10.3±3.6) mm, P=0.001], higher TBUT [(15.0±4.2) vs (10.2±3.6) s, P=0.003], lower fluorescein staining score [0 (0, 1) vs 2 (1, 4), P<0.001], and higher goblet cell density at conjunctival fornix (27.2±7.6 vs 10.7±4.8, P<0.001). Additionally, compared with the model group, the conjunctiva of the treatment group expressed a significantly lower level of miRNA-744-5p (1.67±0.24 vs 2.63±0.58, P<0.001) and interleukin-6 [2.38 (1.84, 4.61) vs 4.82 (3.99, 6.36), P=0.022]. Meanwhile, the treatment group showed significantly increased level of 16∶1, Δ9 fatty acid [(10.31±1.00)% vs (3.87±0.45)%, P<0.001] and iso-18∶0 fatty acid [(7.09±0.93)% vs (2.44±0.70)%, P<0.001], but decreased level of iso-26∶0 fatty acid [(5.72±1.07)% vs (8.02±0.65)%, P<0.001] in the meibum compared with the model group. Conclusion: Androgen replacement therapy can alleviate dry eye signs in rabbits presented with combined androgen deficiency and MGD.