Novel therapeutic targets are required for the treatment of Parkinson's disease (PD). Previous studies suggest that the Rho/Rho‑associated, coiled‑coil‑containing protein kinases (ROCKs) signaling pathway may be a promising therapeutic target in PD. To elucidate the importance of ROCKII in the pathogenesis of dopaminergic (DA) neuron loss and to investigate the efficacy of ROCK inhibitors in PD, ROCKII expression in the substantia nigra (SN) of mice was silenced through the injection of a lentivirus‑based small hairpin RNA system. Empty lentivirus vectors served as controls. Mice were subsequently challenged with 1‑methyl‑4‑phenyl‑1,2,3,6‑tetrahydropyridine (MPTP). The expression levels and activity of ROCKII were elevated in tyrosine hydroxylase‑positive neurons and in cluster of differentiation (CD) 11b‑positive microglia within the SN of MPTP‑treated mice, which was accompanied by an increased level of expression of inducible nitric oxide synthase (iNOS) and activation of the Toll‑like receptor (TLR)2/nuclear factor (NF)‑κB signaling pathway in M1 microglia. ROCKII interference (RI) significantly improved movement disorder and attenuated DA neuron loss induced by MPTP. In addition, RI inhibited the activation of M1 microglia in the SN, exhibiting reduced activity of the TLR2/NF‑κB signaling pathway and decreased expression levels of iNOS and inflammatory factors, including interleukin (IL)‑1β and IL‑6. The results of the present study verify that ROCKII participates in the loss of DA neurons induced by MPTP and suggest that ROCKII inhibition may be a promising therapeutic target for PD.