Interleukin‐33 (IL‐33), a member of the IL1 family, has been found to be expressed in oligodendrocytes (OLGs) and released as an alarmin from injured OLGs to work on other glial cell‐types in the central nervous system. However, its functional role in OLGs remains unclear. Herein, we present that IL‐33 was mainly expressed in the nucleus of CC1+‐oligodendrocytes (OLGs) in mouse and rat corpus callosum, as well as NG2+‐oligodendrocyte precursor cells (OPCs). The in vitro study indicated that the amount of IL‐33 expressing in OPCs was higher when compared to that detected in OLGs. Results from the experiments using lentivirus‐mediated shRNA delivery against IL‐33 expression (IL33‐KD) in OPCs showed that IL33‐KD reduced the differentiation of OLGs into mature OLGs along with the down‐regulation of OLG differentiation‐related genes and mature OLG marker proteins, myelin basic protein (MBP) and proteolipid protein (PLP). Alternatively, we observed reduced differentiation of OLGs that were prepared from the brains of IL‐33 gene knockout (IL33‐KO) mice with anxiolytic‐like behavior. Observations were correlated with the results showing lower levels of MBP and PLP in IL33‐KO cultures than those detected in the control cultures prepared from wildtype (WT) mice. Transmission Electron Microscopy (TEM) analysis revealed that the myelin structures in the corpus callosum of the IL33‐KO mice were impaired compared to those observed in the WT mice. Overall, this study provides important evidence that declined expression of IL‐33 in OPCs suppresses the maturation of OLGs. Moreover, gene deficiency of IL‐33 can disrupt OLG maturation and interfere with myelin compaction. Cover Image for this issue: doi: 10.1111/jnc.14522 Our study demonstrates that declined expression of IL‐33 (IL33‐KD) or IL‐33 gene ablation (IL33‐KO) in oligodendrocyte precursor cells (OPCs) reduced oligodendrocyte (OLG) maturation along with the down‐regulation of Olig2, Myrf, and Sox10, as well as OLG lineage markers (MBP, PLP, and CNPase). The findings also implicate that loss of IL‐33 function in OPCs might impair myelin integrity in the white matter. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14522