Sema3c is specifically expressed in the cardiac outflow tract (OFT) of the developing mouse heart and has been implicated in OFT polarization and great artery formation. However, the regulatory basis underlying its spatially restricted expression remains unclear. To investigate the mechanisms underlying OFT-specific Sema3c expression, we utilized chromatin accessibility data from distinct segments of the developing heart and identified a differentially accessible region as an OFT-specific Sema3c enhancer candidate. Unlike previously characterized Sema3c enhancers, this region is located distal to the transcription start site. Reporter analysis using transgenic mouse embryos demonstrated that this region exhibits transcriptional activity from E8.5 onward and remains specifically active in the OFT myocardium throughout heart development. We further defined a minimal 603 bp enhancer whose activity depends on GATA binding sites. This enhancer provides insight into the mechanisms underlying spatially restricted Sema3c expression involved in OFT development.