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 ran?domly 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.