Background Nonselective muscarinic receptor antagonist, atropine, was believed to inhibit myopic progression. The purpose of this study was to determine the efficacy, through topical administration, of the M1-selective muscarinic antagonist pirenzepine in preventing experimentally induced form-deprivation myopia in guinea pigs.Methods Fifty-three guinea pigs, which underwent monocular deprivation with their eyelids sutured, were divided into 6 groups. Three groups were treated with 1%, 2% or 4% pirenzepine ophthalmic solutions; the fourth group with atropine; the fifth with saline and the last group left untreated. Ocular refraction, in vivo biometric measurements and wet eye weight were collected before and after the experiment. All the eyes were finally enucleated for histopathological examination to evaluate the possible toxic effects on ocular structures.Results Animals untreated or treated with saline produced (-2.31±1.47) D and (-2.25±0.88) D of axial myopia respectively. Those treated with 1% pirenzepine ophthalmic solution produced relative myopia of (-1.63±0.48) D, and those under the treatment of 2% and 4% pirenzepine ophthalmic solution only developed a relative myopia of (-0.89±0.42) D and (-0.70±0.41) D (F=9.56, P<0.05). The significant reduction in myopia in 2% and 4% pirenzepine treated animals was caused by significantly less vitreous chamber elongation and axial elongation of the deprived eyes [2% group: (0.009±0.052) mm, 4% group: (0.006±0.078) mm] when compared with untreated, saline treated or 1% pirenzepine treated guinea pigs [(0.057±0.056) mm, (0.064±0.053) mm and (0.033±0.035) mm, respectively]. Histological examinations revealed no obviously toxic effects on the eyes treated with pirenzepine.Conclusion Topical administration of the M1-selective muscarinic antagonist, pirenzepine, can prevent induced form-deprivation myopia in guinea pigs by inhibiting axial elongation without obvious damage to ocular tissues.