The longitudinal twist represents an intrinsic morphological characteristic of native cellulose microfibrils, which was generated during the isolation and drying processes. The molecular underpinnings of the right-handed chirality exhibited by this twist have been extensively investigated through molecular dynamics simulations and density functional theory calculations. Various mechanisms have been proposed to explain this phenomenon, with some attributing it to van der Waals (vdW) interactions, intra- or inter-chain hydrogen bonds, or the anisotropy between hydrogen bonding (HB) and vdW interactions, while others have refuted these propositions. This divergence in explanations has fueled an ongoing debate regarding the origin of the twist. Our research has uncovered that allomorphic conversion from cellulose I to II can modulate the chirality from right-handed to left-handed. Further analysis has illuminated that antiparallel glucose stacking is responsible for inducing left-handed chirality, whereas parallel stacking leads to right-handed chirality. Additionally, both the glycosidic linkage and the six-membered pyranose ring exhibit flexibility and collectively determine the chirality of the cellulose microfibrils.