Collectively, the present study demonstrated that GSK-J4 treatment effectively suppressed osteogenic differentiation of Twist-1del/+ calvarial cells in both sexes and whole calvaria explants in vitro, and prevented coronal suture craniosynostosis of Twist-1del/+ mice in vivo by inhibiting the enzymatic activity of aberrant Kdm6a and Kdm6b levels and thus recovering the level of H3K27me3 marks on osteogenic genes. This evidence concerns the gene KDM6B and craniosynostosis.