Purpose: To investigate the effects of intravitreal injection of cyclopentolate on form deprivation myopia in guinea pigs. Methods: Thirty-five guinea pigs at age of 3 weeks were randomly divided into 5 groups (n = 7 for each group): deprived, deprived plus saline, deprived plus cyclopentolate, normal control, and cyclopentolate group. Form deprivation was only performed in right eyes with translucent membranes for 4 weeks. Physiological saline and cyclopentolate were intravitreally injected into deprived eyes at four-day intervals. All the left eyes remained untreated as group control. Refraction was measured by retinoscopy after cycloplegia. The axial dimensions were measured by A-scan ultrasound. Subsequently, retinal histology was observed by light microscopy. Results: After 4 weeks of treatment, intravitreal injection of cyclopentolate significantly reduced the degree of myopia in the deprived eyes (from -3.92 D to -0.86 D, P < 0.001), and retarded the increase of vitreous chamber depth (from 3.83 ± 0.06 mm to 3.70 ± 0.05 mm, P < 0.001) and axial length (from 8.42 ± 0.04 mm to 8.30 ± 0.05 mm, P < 0.001) in the deprived eyes. Histological ex-amination revealed no evidence of retinal damage of eyes injected with physiological saline or cy-clopentolate compared with normal control eyes. Conclusions: Intravitreal administration of cy-clopentolate reduces axial elongation of the deprived eyes in guinea pigs. Further investigations are required to identify the optimal dose.