AbstractDirect laser writing (DLW) via two‐photon polymerization is an emerging highly precise technique for the fabrication of intricate cellular scaffolds. Despite recent progress in using two‐photon‐polymerized scaffolds to probe fundamental cell behaviors, new methods to direct and modulate microscale cell alignment and selective cell adhesion using two‐photon‐polymerized microstructures are of keen interest. Here, a DLW‐fabricated 2D and 3D hydrogel microstructures, with alternating soft and stiff regions, for precisely controlled cell alignment are reported. The use of both cell‐adhesive and cell‐repellent hydrogels allows selective adhesion and alignment of human mesenchymal stem cells within the printed structure. Importantly, DLW patterning enables cell alignment on flat surfaces as well as irregular and curved 3D microstructures, which are otherwise challenging to pattern with cells.