Complete and specific ablation of a single dopaminergic (DA) pathway is a critical step to distinguish the roles of DA pathways in vivo. However, this kind of technique has not been reported in non-human primates. This study aimed to establish a lesioning method with a complete and specific ablation. A carefully designed infusion route based on a MRI stereotactic technique was developed to deliver the highly selective dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP+) unilaterally into multiple sites of compact part of substantia nigra (SNc) and striatum in monkeys. The nigrostriatal DA pathway was selected because lesioning of this pathway may induce symptoms that are suitable for evaluation. The pathological, behavioral, neuropharmacological, and clinical laboratorial data were collected to evaluate the lesioning effects. Pathological examination revealed a complete ablation of tyrosine hydroxylase positive (TH+) neurons in the SNc, while preserving intact TH+ neurons in the ventral tegmental area (VTA) nearby. TH+ projections in the striatum were also unilaterally lost. The monkeys displayed stable (>28 weeks) rotations and symptoms which were expected with loss of DA neurons in the SNc, with rest tremor being an exception. No item implied the presence of a severe side effect caused by the operation or the intracerebral MPP+ infusion. The results suggested that rest tremor may not directly rely on the nigrostriatal pathway. Taken together, in addition to providing a specific nigrostriatal DA lesioned model, this method, combined with brain stimulation or other techniques, can be applied as a powerful tool for the complete lesion of any desired DA pathway in order to study its specific functions in the brain.