Cruciform structures of DNA occur in sequences exhibiting an inverted repeat (IR) structure. They are known to play a role in several regulatory processes of cellular physiology, and have been linked to the development of diseases such as Werner’s syndrome and certain types of cancer (1). Magnetic tweezer assays have enabled the study of these non-canonical DNA structures on a single-molecule scale (2).However, a detailed understanding of structural and kinetic properties of DNA cruciforms is still lacking. We used a coarse-grained computational model of DNA (3) to study thermodynamic stability and formation kinetics of cruciform structures in a superhelically stressed DNA strand at physiological temperatures. Thermal quantities of the structures in the DNA model were obtained using a cluster-move Monte Carlo (MC) method. Free-energy landscapes governing the cruciform formation process in a 34bp IR sequence were calculated using umbrella sampling. Kinetic properties of cruciform formation were studied in direct, unbiased MC simulations for 34bp and 64 bp IR sequences. In the simulations of the cruciform structures we find an asynchronous, but cooperative extrusion mechanism (4). First, a bubble has to diffuse to the centre position of the IR sequence. Then, a size fluctuation of the bubble must be large enough to allow for rearrangement of its single strands, leading to formation of a first hairpin. Upon formation of the first cruciform arm, the second arm rapidly grows to a similar size. This initial asynchronous formation is followed by a synchronous growth mechanism of both cruciform arms, which resolves the remaining superhelical stress. The asynchronous formation mechanism is reflected in the free energy landscapes of the system, which we calculated as a function of the number of base pairs in the system. For all parameters studied, the free energy barrier lies at 8-9kBT. At a temperature of 39.4°C, we observe that the fully extruded cruciform structure is thermodynamically more stable than the corresponding bubble state of the DNA strand. This result consistent with the in vivo genetic instability observed for perfect IR sequences (2). We have used numerical simulations of a coarse-grained model of DNA to study thermodynamic and structural properties of DNA cruciforms at a length and time resolution presently inaccessible to experimental setups. Our results show a complex asynchronous extrusion behaviour driven by DNA supercoiling and ambient temperature. The genetic instability of perfect IR sequences may be due to the high thermodynamic stability of cruciform structures under physiological conditions of temperature and supercoiling. This highlights the importance of physical properties of the DNA heteropolymer in physiological processes on a molecular scale, such as cruciform extrusion.