Mechanisms of the assembly of actin stress fibers (SFs) have been extensively studied, while those of the disassembly-particularly cell shortening-induced ones-remain unclear. Here, we show that SFs have helical structures composed of multi-subbundles, and they tend to be delaminated upon cell shortening. Specifically, we observed with atomic force microscopy delamination of helical SFs into their subbundles. We physically caught individual SFs using a pair of glass needles to observe rotational deformations during stretching as well as ATP-driven active contraction, suggesting that they deform in a manner reflecting their intrinsic helical structure. A minimal analytical model was then developed based on the Frenet-Serret formulas with force-strain measurement data to suggest that helical SFs can be delaminated into the constituent subbundles upon axial shortening. Given that SFs are large molecular clusters that bear cellular tension but must promptly disassemble upon loss of the tension, the resulting increase in their surface area due to the shortening-induced delamination may facilitate interaction with surrounding molecules to aid subsequent disintegration. Thus, our results suggest a new mechanism of the disassembly that occurs only in the specific SFs exposed to forced shortening.