Deposition of mussel-inspired polydopamine (PDA) has rapidly emerged as a simple yet effective strategy to functionalize the surface of biomaterials. The experimental simplicity of the deposition process, combined with native bioactivity and bioadhesive properties, make PDA an attractive solution for biomedical applications, ranging from functional biomaterials for tissue engineering to antibacterial surfaces. Unveiling the interplay among deposition parameters, physicochemical properties of the resulting structures and their functions, is a fundamental aspect to unlock a more sophisticated knowledge of PDA biofunctionalization and its role in controlling key biological events, such as stem cell response. Although the mechanism for the bioinductive capacity of PDA is not fully understood, surface topography, chemistry and adhesive properties are believed to play a critical role, either individually or in combination. This work addresses the differential roles of such surface properties on PDA bioactivity. We achieved novel insights on the physicochemical makeup of two PDA coatings obtained by varying one critical parameter (i.e., solution agitation) during the deposition. Successively, we focused on the effects on human mesenchymal stem cells (hMSCs) in both normal and serum-free culturing conditions. This study reveals both the serum-dependent and independent cueing involved in bioactive induction caused by PDA.