Three-dimensional (3D) genome is essential in biological processes such as gene transcription regulation and neurodevelopment. SETDB1 is the histone H3 lysine 9 methyltransferase, which involved in histone modification and 3D genome remodeling. In this study, mouse Setdb1 was specifically knocked out in neural precursor cells (NPCs), resulting in a significant increase of compartment counts in the 3D genome, A and B compartments switched, the cis interactions between A and B compartments were significantly reduced, while the cis interactions between A and A compartments, and between B and B compartments were significantly increased. SETDB1 is the H3K9me3’s writer，after specifically knocking out Setdb1 in NPCs, the ChIP-seq of H3K9me3 showed that, the enrichment of H3K9me3 peaks in euchromatin was down, particularly, we found that the H3K9me3’s up peaks were enriched in heterochromatin. HP1α can interact with Setdb1-KAP1-KRAB-ZNF complex, as the H3K9me3’s reader, together promote the modification of  H3K9me3’s inhibitory, after specifically knocking out Setdb1 in NPCs, the same thing happened to the ChIP-seq of the HP1α. Thus, the hypothesis of this study is that the absence of SETDB1 breaks the restriction of H3K9me3 to HP1α in euchromatin, since HP1α has been reported to generate phase separation on its own, it is possible that free HP1α is recruited by phase separation in heterochromatin and forms complexes with histone methyltransferases in heterochromatin to mediate the formation of repressive histone modification -H3K9me3 then affect 3D genome structure. This result can help us to understand the epigenetic molecular mechanism of SETDB1 in regulating 3D genome structure from the perspective of multi-factor regulation.