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The topographical micro-patterning of nanofibrillar collagen gels is promising for the fabrication of biofunctional constructs mimicking topographical cell microenvironments of in vivo extracellular matrices. Nevertheless, obtaining structurally robust collagen micro-patterns through this technique is still a challenging issue. Here, we report a novel in situ photochemical crosslinking-assisted collagen embossing (IPC-CE) process as an integrative fabrication technique based on collagen compression-based embossing and UV–riboflavin crosslinking. The IPC-CE process using a micro-patterned polydimethylsiloxane (PDMS) master mold enables the compaction of collagen nanofibrils into micro-cavities of the mold and the simultaneous occurrence of riboflavin-mediated photochemical reactions among the nanofibrils, resulting in a robust micro-patterned collagen construct. The micro-patterned collagen construct fabricated through the IPC-CE showed a remarkable mechanical resistivity against rehydration and manual handling, which could not be achieved through the conventional collagen compression-based embossing alone. Micro-patterns of various sizes (minimum feature size <10 μm) and shapes could be obtained by controlling the compressive pressure (115 kPa) and the UV dose (3.00 J/cm²) applied during the process. NIH 3T3 cell culture on the micro-patterned collagen construct finally demonstrated its practical applicability in biological applications, showing a notable effect of anisotropic topography on cells in comparison with the conventional construct. Full article