Abstract: Slitrk3, a neurosynaptic-related protein, can regulate the development of inhibitory synapses. To explore the transcriptional regulation mechanism of slitrk3 gene of the large yellow croaker (Larimichthys crocea) can provide a new idea for solving the problems of growth, stress and anti-stress in the L. crocea culture. We conducted a multiple sequence alignment and a phylogenetic tree analysis of amino acids for Slitrk3 in L. crocea and other species to investigate the transcriptional regulation mechanism of the neural cell adhesion molecule for slitrk3 gene. Besides, we predicted the potential core promoter regions, CpG islands and transcription factor binding sites of slitrk3 gene by bioinformatics methods, and detected the Luciferase activity of promoter of slitrk3 gene by the Dual-Luciferase Reporter System. Bioinformatics analysis shows that the amino acid sequences of Slitrk3 were highly conserved in fish. There were two transcription start sites, two CpG islands, and multiple transcription factor binding sites such as Sp1, GR, C/EBPα and C/EBPβ in promoter of slitrk3 gene. Dual-Luciferase Reporter System shows that the regions from −1970 to −1614 bp and from −1 210 to −667 bp contained positive regulatory elements; while the regions from −1614 to −1210 bp, from −667 to −376 bp and from −376 to −147 contained negative regulatory elements; and the regions from −147 to +16 bp might be core promoter of slitrk3 gene. The results lay a theoretical foundation for the further study of transcriptional regulation mechanism of slitrk3 gene in L. crocea.