Heterochromatin is a key architectural feature of eukaryotic chromosomes, which is critical for cell-type specific gene expression and genome stability. In the mammalian nucleus, heterochromatin is segregated from transcriptionally active genomic regions and exists as large condensed and inactive nuclear compartment. However, the underlying mechanism of spatial organization of heterochromatin is still poorly understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that define constitutive and facultative heterochromatin, respectively. In mammals, there are at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization by using a combination of mutant cells for the five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We show that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation, and loss of both H3K9 and H3K27 methylation resulted in impaired both condensation and spatial organization of heterochromatin. Our data demonstrate that heterochromatin is plastic and the two major repressive epigenome pathways exclusively but also redundantly maintain H3K9me2/3-marked heterochromatin organization in mammalian cells.