The topographic features of an implant, which mechanically regulate cell behaviors and functions, are critical for the clinical success in tissue regeneration. How cells sense and respond to the topographical cues, e.g., interfacial roughness, is yet to be fully understood and even debatable. Here, the mechanotransduction and fate determination of human mesenchymal stem cells (MSCs) on surface roughness gradients are systematically studied. The broad range of topographical scales and high‐throughput imaging is achieved based on a catecholic polyglycerol coating fabricated by a one‐step‐tilted dip‐coating approach. It is revealed that the adhesion of MSCs is biphasically regulated by interfacial roughness. The cell mechanotransduction is investigated from focal adhesion to transcriptional activity, which explains that cellular response to interfacial roughness undergoes a direct force‐dependent mechanism. Moreover, the optimized roughness for promoting cell fate specification is explored.