Objective To evaluate the inhibitory effect of highly selective M4 receptor antagonist MT3 on the form deprivation myopia in guinea pigs and its potential mechanism. Methods Thirty-two healthy male guinea pigs were randomly divided into three groups: control group, form deprivation group, and form deprivation+MT3 group, 8 animals in each group. Refraction was measured by retinoscopy after cycloplegia before and after the experiment. The ocular biological dimensions were measured by A-scan ultrasound. RT-PCR was used to detect the relative expression of TGF-β2 mRNA in the retina and choroid.Results Compared with the right eyes of control group, the right eyes of form deprivation+MT3 group developed relative myopia of -1.44±0.50 D (right-left eye) (P=0.001). The vitreous chamber depth and axial length of the right eyes were significantly prolonged by 0.10±0.02 mm and 0.14±0.07 mm (P<0.001, P<0.001), respectively, but the increases of myopia and axial length were significantly smaller than that of the form deprivation group (P<0.001, P<0.001, P<0.001). Down-regulation of relative mRNA expression of TGF-β2 in retina and choroid was found in the form deprivation group (P<0.001, P=0.014) compared with the right eyes of the control group, while up-regulation of relative mRNA expression of TGF-β2 in retina and choroid was found in the form deprivation+MT3 group (P<0.001, P<0.001). Conclusions MT3 can inhibit the development of form deprivation myopia in guinea pigs, which may play an important role by the regulation of TGF-β2 mRNA level in the retina and choroid.