Background: Diffuse intrinsic pontine glioma (DIPG) is a rare and fatal pediatric brain cancer without cure. Seeking therapeutic strategies is still a major challenge in DIPG research. Previous study has shown that dysregulation of G1/S cell cycle checkpoint was common in DIPG and this dysregulation is even more enriched in the H3.3K27 M mutant subgroup. Here we assess potential anti-tumor efficacy of palbociclib, a specific and cytostalic inhibitor of CDK4/6, on high grade H3.3-K27 M-mutant DIPGs in vitro and in vivo. Methods: We established patient-derived cell lines from treatment-naive specimens. All the lines have H3.3K27 M mutation. We used a range of biological in vitro assays to assess the effect of palbociclib on growth of DIPGs. Palbociclib activity was also assayed in vivo against three independent DIPG orthotropic xenografts model. Findings: Dysregulation of G1/S cell cycle checkpoint is enriched in these DIPGs. Then, we showed that depletion of CDK4 or CDK6 inhibits DIPG cells growth and blocks G1/S transition. Furthermore, palbocidib effectively repressed DIPG growth in vitro. Transcriptome analysis showed that palbociclib not only blocks G1/S transition, it also blocks other oncogenic targets such as MYC. Finally, palbocidib activity was assayed in vivo against DIPG orthotropic xenografts to demonstrate the high efficiency of blocking tumor growth. Interpretation: Our findings thus revealed that palbocidib could be the therapeutic strategy for treatment-naive DIPG with H3.3K27 M mutation. (C) 2019 The Authors. Published by Elsevier B.V.