Great advantages bestowed by mesoporous silica nanoparticles (MSNs) including high surface area, tailorable pore diameter and surface chemistry, and large pore volume render them as efficient tools in biomedical applications. Herein, MSNs with different surface chemistries were synthesized and investigated in terms of biocompatibility and their impact on the morphology of bone marrow-derived mesenchymal stem cells both in 2D and 3D culture systems. Bare MSNs (BMSNs) were synthesized by template removing method using tetraethylorthosilicate (TEOS) as a precursor. The as-prepared BMSNs were then used to prepare amine-functionalized (AMSNs), carboxyl-functionalized (CMSNs) and polymeric amine-functionalized (PMSNs) samples, consecutively. These nanoparticles were characterized by scanning electron microscopy, zeta potential measurement, dynamic light scattering, BET (Brunauer, Emmett, Teller) analysis, and FTIR technique. In a 3D culture system, stem cells were encapsulated in alginate hydrogel in which MSNs of different functionalities were incorporated. The results showed good biocompatibility for both BMSNs and AMSNs in 2D and 3D culture systems. For these samples, the viability of about 80% was acquired after 2 weeks of 3D culture. When compared to the control, CMSNs caused higher cell proliferation in the 2D culture; while they showed cytotoxic effects in the 3D culture system. Interestingly, polymeric amine-functionalized silica nanoparticles (PMSNs) resulted in disrupted morphology and very low viability in the 2D cell culture and even less viability in 3D environment in comparison to BMSNs and AMSNs. This significant decrease in cell viability was attributed to the higher uptake values of highly positively charged PMSNs by cells as compared to other MSNs. This up-regulated uptake was evaluated by using an inductively coupled plasma optical emission spectroscopy instrument (ICP-OES). These results uncover different interactions between cell and nanoparticles with various surface chemistries. Building on these results, new windows are opened for employing biocompatible nanoparticles such as BMSNs and AMSNs, even at high concentrations, as potential cargos for carrying required growth and/or differentiation factors for tissue engineering applications.