Cerebral ischemic injury is a major cause of death and long‑term disability worldwide that leads to neurological and behavioral deficits, and for which successful treatments are still lacking. Ras homolog family member A (RhoA) and Rho‑associated coiled‑coil containing protein kinase (ROCK) are associated with the growth of neurons and the movement of neuronal growth cones. RhoA/ROCK inhibitors have been demonstrated to promote the recovery of motor function following nerve injury, but the underlying mechanism requires further investigation. The present study aimed to investigate the effects of the ROCK inhibitor Y‑27632 on middle cerebral artery occlusion (MCAO)‑induced cerebral ischemic injury. Rats were randomly assigned to the Control, Y‑27632, MCAO + Vehicle or MCAO + Y‑27632 group. Firstly, infarct volume, cognitive ability and cerebral injury were assessed. Secondly, indicators of cerebral inflammation, oxidative stress and apoptosis were evaluated. Finally, the expression of recombinant glial fibrillary acidic protein (GFAP) and allograft inflammatory factor 1 (AIF1) in the brain were measured to assess the activation of astrocytes and microglia, respectively. The results showed that Y‑27632 effectively increased the survival rate and behavioral performance of rats, and attenuated the cerebral injury, oxidative stress and cerebral inflammation levels following MCAO. The disturbance in hippocampal neurons caused by MCAO was also alleviated following treatment with Y‑27632. Neuronal apoptosis was also decreased following Y‑27632 treatment, as demonstrated by the TUNEL assay and the expression levels of Caspases‑3, 8 and 9 and Bax/Bcl‑2 ratio. The levels of GFAP and AIF1 were increased by MCAO and further promoted by Y‑27632, indicating the activation of astrocytes and microglia. In conclusion, the present study offered evidence of a protective effect of Y‑27632 administration on cerebral ischemia/reperfusion induced behavioral and hippocampal damage by activating astrocytes and microglia.