Chromatin is the substrate for all DNA-associated processes; yet the mechanisms regulating its structure and dynamics remain elusive. Nucleosome positioning patterns depend on sequence and vary genome-wide: inactive heterochromatic domains have regularly positioned nucleosome arrays, in contrast with the variable positioning of active chromatin. Here we show this positioning determines 3D chromatin structure, as well as its mechanical response and dynamics. Irregular nucleosome arrays form heterogeneous and decondensed structures, which are are more fragile under tension than regularly spaced arrays; they also display a much more dynamic sampling of structures. These findings indicate that intrinsic nucleosome regularity may underpin the structural stability of heterochromatin, whilst irregular positioning provides dynamic plasticity to open and remodel euchromatin. These results reconcile major discrepancies between observations of ordered structures in studies with repetitive sequences, and recent evidence for heterogeneous chromatin in living cells.