The relationship between microstructure and mechanical properties has been investigated in Argiope trifasciata dragline silk fibers (major ampullate silk, MAS) by X-ray diffraction, Raman spectroscopy and tensile testing. We have analyzed three fractions of the material, i.e. amorphous, highly oriented nanocrystals and weakly oriented material, for different values of the macroscopic alignment parameter α, calculated as the relative difference between the length of the fiber and its length when supercontracted. Two distinct regimes have been identified: for low values of the alignment parameter α, microstructural changes are dominated by the reorientation of the nanocrystals; however, at high values (α > 0.5) of the alignment parameter, an increase in the fraction of the crystalline phase is revealed. The two regimes are also reflected in the mechanical behaviour, which can be explained by microstructural changes. This finding of the two distinct regimes in the microstructural evolution, which separates the reorientation and the increase in the crystalline phase, will be valuable to develop and validate molecular models of natural and artificial silk fibers, as well as to deepen our present knowledge of the origin of the outstanding properties of MAS fibers. In addition, we have analyzed the characteristics of the crystal lattice, and discussed the relationship between the percentage of short side-chain residues and the unit cell dimensions in different silks.