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AIM To investigate the effects of micro-injection of botulinum toxin A (BTXA) on acute acquired comitant esotropia (AACE). METHODS A total of 33 AACE patients who underwent BTXA micro-injection at Renmin Hospital of Wuhan University from September 1st, 2019 to July 1st, 2021 were retrospective analyzed. Esotropia, eye alignment, stereopsis, and complications were examined at baseline (except complications), 1wk, 1, 3, and 6mo after injection. RESULTS The average angle of deviation before injection was (+20.24±6.80)Δ at near and (+24.76±6.43)Δ at distance, while (+5.15±5.85)Δ at near and (+7.30±6.17)Δ at distance 6mo after treatment (P<0.05). Six months after injection, the stereopsis of patients had improved. The number of patients having no stereopsis (>800 seconds of arc) decreased from 11 to 3. The number of patients having peripheral stereopsis (300-800 seconds of arc), macular stereopsis (70-200 seconds of arc) and central concave stereopsis (≤60 seconds of arc) increased from 10 to 11, 10 to 12, and 2 to 7, respectively. At the follow-ups at 1wk, 1, 3, and 6mo after injection, success rates were 96.97%, 96.97%, 93.94% and 87.88%, respectively. One week after injection, two patients (6.07%) showed subconjunctival hemorrhage; three patients (9.09%) showed limited eye movement and one patient (3.03%) showed mild vertical strabismus. All the symptoms disappeared by the final follow-up. CONCLUSION Micro-injection of BTXA can reduce diplopia and improve binocular vision function of AACE patients. Furthermore, the operation is relatively safe with few complications, making it an ideal treatment modality for AACE.