Cell chirality plays a critical role in the linkage between molecular chirality and the asymmetrical biological functions of body organs. However, enantioselective interactions between cell chirality and the extracellular environment are not yet fully understood. In this study, we investigated the effects of structurally chiral extracellular microenvironments on cellular alignments and differentiations. Twisted wrinkle-shaped chiral micropatterns were prepared using biaxial and asymmetric buckling methods, wherein structural handedness was determined from the orientation of the tilt angle between the first and second microwrinkles. Myoblasts were separately cultured on two enantiomeric chiral micropatterns in a mirror-reflected shape. Cells cultured on the left-handed chiral micropatterns preferred alignments along the direction of the second microwrinkle, with a relatively deeper valley than that of the first microwrinkle. The aligned cells on the left-handed pattern showed higher differentiation rates, as assessed by fusion indices and marker protein expression levels, than those cultured on right-handed chiral micropatterns. These results suggest that myoblasts exhibit enantioselective recognition of structurally chiral microenvironments, which can promote cellular alignments and differentiation.