X-linked intellectual disability (XLID) is a well-known form of intellectual disability which is specifically associated with X-linked recessive inheritance. To investigate the function of XLID gene, ZC4H2, we generated zebrafish mutant for ZC4H2 homologue gene using TALEN methodology. Zebrafish zc4h2 homozygous mutant larvae exhibited abnormal swimming, stationary eye movement, and hyperactivity of jaw and pectoral fin. Because of the behavioral defects were consistent with hyperactivity, we focused on underlying neuronal defects. Through various molecular markers, we observed a striking reduction in GABAergic interneurons. Analysis of cell type specific markers showed a specific loss of V2 interneurons in the midbrain tegmentum and spinal cord. Also, we found that loss of Zc4h2 function affects in misexpression of dbx2/nkx6.1 boundary. To restore the Zc4h2 function in the mutant, we microinjected human and zebrafish mRNAs in zc4h2 mutant embryos, and examined gad1b-positive neurons. As a result, human and zebrafish mRNAs rescued the mutant phenotype. However, mutant zebrafish microinjected with human p.L66H or p.R213W mRNA failed to be rescued, while the p.R18K mRNA was able to rescue the interneuron defect. In this studies clearly support ZC4H2 as a novel XLID gene with a required function in V2 interneuron development. Loss of function of ZC4H2 thus likely results in altered connectivity of many brain and spinal circuits.