Objective: The aim of this study was to evaluate the therapeutic utility of sirolimus liposomes (prepared by two different techniques at two different doses) by subconjunctival injection, and tacrolimus ophthalmic solution at 0.03% in canine patients diagnosed with keratoconjunctivitis sicca (KCS) non-responsive to conventional treatment. Procedures: 20 privately owned pet dogs with clinically confirmed non-responsive KCS were randomized into 5 groups. Patients were administered with 0.15 ml of placebo or product respectively every 2 weeks for 60 days. Also, all patients received topically tacrolimus 0.03% solution 3 times a day for 90 days. Each animal was given a full ophthalmic examination prior and during the evaluation. Results: During the evaluation, no adverse effects were observed. For a 0.16 mg/ml dose of sirolimus, the heating method provided liposomes with enhanced immunomodulating activity in contrast to the ethanol injection method. On the other hand, the improvement in lacrimal production achieved by a 0.4 mg/ml sirolimus dose was independent of the preparation technique. HM4 and EI16 formulations showed important decrease in secondary pigmentary keratitis. Also, the use of sirolimus liposomes exhibited reduction in corneal vascularization and conjunctival discharge. Conclusions: The 0.4 mg/ml dose provided greater improvement of lacrimal production and minimization of ocular irritation indicators with no influence of the preparation technique used. The heating method generated liposomal dispersions with enhanced immunomodulating activity at a lower dose of 0.16 mg/ml. Besides lacrimal production improvement, the treatment significantly improved patient vision clarity.