Purpose: To report the outcomes of bupivacaine injection for the treatment of comitant horizontal strabismus and evaluate clinical effectiveness and associated radiological changes. Methods: This prospective observational clinical study was conducted on 10 patients with comitant horizontal strabismus of up to 40 prism diopters. Ophthalmologic examinations and three-dimensional orbital magnetic resonance imaging were performed pre and post-injection (at first, third, and 12th months). A 4.5 ml of 0.5% bupivacaine was injected into the extraocular muscle under topical anesthesia using an electromyography in all patients. Results: The mean follow-up time at post bupivacaine injection and mean deviation at primary position were was 17 +/- 2 months and 21.3 prism diopters, respectively. The mean changes in ocular alignment, enlargement of the cross-sectional area in the injected muscle, and volumetric enlargement were 7.7 PD, 12%, and 17% at the first year post-injection, respectively. No severe or persistent complication was observed. Ptosis and mydriasis were noted post-injection due to the anesthetic effects of bupivacaine but disappeared within 2 h post-injection. Conclusions: Bupivacaine injection improved eye alignment in small-angle horizontal comitant strabismus, effectively diagnosed with orbital magnetic resonance imaging to evaluate volumetric changes of the extraocular muscles. Further clinical studies with larger numbers of patients should be performed to define optimal dosages, concentration, and application method and dose-response relationship.