Objective To investigate the effect of the placenta-derived three-dimensional (3-D) spheroid-cultured mesenchymal stem cells (MSCs) on the expression of tumor necrosis factor-α (TNF-α) and apoptosis-related proteins in rats with cerebral ischemia-reperfusion injury, so as to explore the neuroprotective effects and mechanism of 3-D spheroid-cultured MSCs. Methods The experimental animals were randomly divided into Sham group, Vehicle group and 3-D spheroid-cultured MSCs treated group (n=36). In the Sham group, the middle cerebral artery (MCA) was simply isolated but not ligated. In the MSC treated group, focal ischemia/reperfusion in rats was induced by intraluminal middle cerebral artery occlusion (MCAO) with a nylon monofilament suture and then MSCs transplantation was done. In the Vehicle group, the MCAO model was set up and an equal volume of vehicle was given. Neurological scores of animals were evaluated at different time points: the first day, the third day and the seventh day after surgery. Enzyme-linked immunosorbent assay (ELISA) and RT-PCR method were used to detect TNF-α expression in rat brain tissues. The SABC immunohistochemical staining method was used to detect the expression of Caspase-3 and Caspase-8 in rat brain tissues. Results The neuromotor function of rats in MSCs treated group was significantly decreased compared with those in the other two groups (P <0.05). Compared with the Sham group, the expressions of TNF-α, Caspase-3 and Caspase-8 in the brain tissue were significantly increased in the Vehicle group (P <0.01). Moreover, compared with the Vehicle group, expression of TNF-α, Caspase-3 and Caspase-8 in the brain tissue was significantly decreased in MSCs treated group (P <0.05, P <0.01). Conclusion 3-D spheroid-cultured MSCs transplantation may improve the neuromotor function in rats with cerebral ischemia/reperfusion injury via down-regulating inflammatory cytokine TNF-α and expression of Caspase-3 and Caspase-8 in the brain tissue.