In higher eukaryotes, heterochromatin is mainly composed of transposable elements (TEs) silenced by epigenetic mechanisms. But, the silencing of certain heterochromatin-associated TEs is disrupted by heat stress. By comparing genome-wide high-resolution chromatin packing patterns under normal or heat conditions obtained through Hi-C analysis, we show here that heat stress causes global rearrangement of the 3D genome in Arabidopsis thaliana. Contacts between pericentromeric regions and distal chromosome arms, as well as proximal intra-chromosomal interactions along the chromosomes, are enhanced. However, interactions within pericentromeres and those between distal intra-chromosomal regions are decreased. Many inter-chromosomal interactions, including those within the KNOT, are also reduced. Furthermore, heat activation of TEs exhibits a high correlation with the reduction of chromosomal interactions involving pericentromeres, the KNOT, the knob, and the upstream and downstream flanking regions of the activated TEs. Together, our results provide insights into the relationship between TE activation and 3D genome reorganization.