Objective To investigate the effect of transplantation with the three-dimensional spheroid-cultured mesenchymal stem cells (MSCs) on the expression of Nogo-A and NgR in rats with cerebral ischemia-reperfusion injury. Methods The experimental animals were randomly divided into Sham group, Vehicle group and MSCs treated group. The model of focal ischemia-reperfusion in rats was induced by intraluminal middle cerebral artery (MCA) occlusion with a nylon monofilament suture in Vehicle group and MSCs treated group. The fishing line was unpluged for reperfusion 2 h after ischemia and MSCs were transplanted in MSCs treated group one day later. Equivalent medium solution was given to the Vehicle group 1 d later. On the 1^st day, 3^rd day, and 7^th day after transplantation, the neuromotor function of the animals was detected. The brain tissue of rats was harvested for RT-PCR detection of Nogo-A and NgR mRNA expression in the brain tissue of rats, and Western blotting analysis was used to detect the expression of Nogo-A and NgR protein. Results Compared with the Vehicle group, the neuromotor function was significantly improved in MSCs treated group on the 7^th day; and the expressions of Nogo-A and NgR mRNA and protein were significantly down-regulated in MSCs treated group on the 1^st day, 3^rd day, and 7^th day after transplantation (P<0.05). Conclusion Transplantation of the three-dimensional spheroid-cultured MSCs can improve the neuromotor function following cerebral ischemia/reperfusion injury, and its mechanism may be associated with down-regulation of Nogo-A and NgR in the brain tissue.