To investigate the effect of Myocardin-related transcription factor A (MRTF-A) on apoptosis induced by ischemic/reperfusion (I/R), middle cerebral artery occlusion /reperfusion (MCAO/R) in rats were applied to mimic I/R. The neurological deficit score, cerebral infarct size, cortical neuron apoptosis and cleaved caspase 3 level were evaluated to determine the effect and the level of apoptosis by TTC straining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) straining, Western blot and immunofluorescence staining. The myeloid cell leukemia-1 (Mcl-1) expression, release of cytochrome C (Cyt C) and its colocalization with apoptotic protease activating factor-1 (Apaf-1) were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining. The results showed that MRTF-A overexpression could decrease the neurological deficit score and reduce cerebral infarct size (P < 0.01 versus Sham). In the MRTF-A-I/R group, TUNEL-positive cells and apoptosis ratio (%) (51.61 ± 6.17%) were significantly decreased compared to the Neg-I/R group (76.45 ± 8.77%) at 24 h reperfusion. Meanwhile, the cleaved caspase 3 expression revealed a similar trend while the expression of Mcl-1 was the opposite. Moreover, MRTF-A overexpression significantly enhanced Mcl-1 fluorescence intensity, which up-regulated the mRNA and protein level (P < 0.05 or P < 0.01 versus Neg-I/R). Furthermore, MRTF-A overexpression markedly inhibited the release of Cyt C, and decreased the colocalization with Apaf-1 in the cytoplasm (P < 0.05 or P < 0.01 versus Neg-I/R). All the data indicated that MRTF-A overexpression could improve the neurological function against cerebral I/R-induced apoptosis since underlying mechanism might be involved in the Mcl-1/Cyt C/cleaved caspase 3 signaling pathway.