SUMOylation has been shown to exert non-negligible influence on transcriptional factors and regulate genes expression by attuning their transcriptional activity. Myocyte-specific enhancer factor-2(MEF2) is a family of transcription factor and play a critical role in embryonic development. The transcriptional activity of MEF2A is highly repressed by SUMOylation in an activity-dependent manner. However, the enzyme that poses de-SUMOylation activity towards MEF2A is still not identified. Here we reported that SENP2 was identified as the major transcriptional dominator and de-SUMOylation enzyme of MEF2A by combining both unbiased shRNA screen and in vivo SUMOylation assays. The SUMOylated form of MEF2A was readily detectable in either SENP2 knockdown cells or knockout embryos. SENP2 markedly enhanced the transcription of MEF2A through directly de-SUMOylation. Moreover, SENP2 protein was accumulated in response to activity-dependent stimuli which in turn mediated activity dependent-regulation of MEF2A de-SUMOylation. Our data clearly show that SENP2 plays an important role in determining the dynamics and functional outcome of MEF2A SUMOylation and transcriptional activation.