To investigate the effect of FK-506 eye drops on Botulinum toxin B (BTX-B)-induced mouse dry eye.Forty-five CBA/J mice were followed up for 4 weeks after treatment with 0.025% FK-506, vehicle or 0.9% saline eye drops 3 days after intralacrimal glands injection with 20 milliunits BTX-B. Tear production, corneal fluorescein staining, the mRNA, and protein expression of cytokines were measured. The activation of nuclear factor-κB (NF-κB) was detected by Western blotting. The infiltration of inflammatory cells was examined by immunohistochemistry.After treated with FK-506 eye drops, aqueous tear production in the mice began to recover at week 1, and then increased to the levels of pre-BTX-B injection at week 4 (2.21 ± 0.43 vs. 2.52 ± 0.71 mm, t = 0.84, P > 0.05). The severity of corneal epithelial defects was alleviated at week 2 and further improved at week 4 when compared with those in the vehicle- and saline-treated groups. The gene expression of IL-1β and TNF-α in the FK-506 and vehicle-treated groups were 47.01% and 45.56%, 85.91% and 115.83% of that in the saline-treated group in the ocular surface, while in the lacrimal glands 49.16% and 67.60%, 94.91% and 95.77% of that in the saline-treated group, respectively. The ratio of phosphorylated IκB-α to total IκB-α in the keratoconjunctival tissues was lower in the FK-506-treated group than in the vehicle- and saline-treated groups (both P < 0.05). No inflammatory cells were detected in all groups.Topical application of FK-506 can inhibit NF-κB activation and related inflammatory response and alleviate the signs of dry eye.Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.