Upregulation of Rho-associated protein kinase 2 (ROCK2) hallmarks the progression of neurodegenerative diseases. However, the molecular mechanisms underlying the regulation of ROCK2 expression are not clear and thus addressed in the current study. We generated a subacute model of Parkinson’s disease in mice with a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) method. The MPTP model was validated by impaired rotational behavior of the mice upon apomorphine exposure, astrocytic activation, and reduction in tyrosine-hydroxylase-positive neurons in the mouse striatum. Moreover, MPTP induced increases in ROCK2 protein but not in ROCK2 mRNA. Further analysis showed that MPTP inhibited the expression of microRNA-291 (miR-291), which suppressed ROCK2 mRNA via 3’-UTR-binding in neuronal cells to increase ROCK2 protein. Intracranial injection of miR-291 four days before MPTP alleviated the impaired rotational behavior of the mice upon apomorphine exposure, MPTP-induced astrocytic activation, and the reduction in tyrosine-hydroxylase-positive neurons in the mouse striatum. Together, these data suggest that miR-291 has a protective role in neurodegeneration, likely through regulation of ROCK2.