Chemically synthesized cross-linkers play decisive roles in variable cargos attached to nanoparticles (NPs). Previous studies reported that surface properties, such as the size, charge, and surface chemistry, are particularly important determinants influencing the biological fate and actions of NPs and cells. Recent studies also focused on the relationship of serum proteins with the surface properties of NPs (also called the protein corona), which is recognized as a key factor in determining the cytotoxicity and biodistribution. However, there is concern that cross-linkers conjugated onto NPs might induce undesirable biological effects. Cell responses induced by cross-linkers have not yet been precisely elucidated. Herein, using mesoporous silica nanoparticles (MSNs) the surfaces of which were separately conjugated with four popular heterobifunctional cross-linkers, i.e., N-[α-maleimidoacetoxy]succinimide ester (AMAS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and maleimide poly(ethylene glycol) succinimidyl carboxymethyl ester (MAL-PEG-SCM), we investigated cross-linker-conjugated MSNs to determine whether they can cause cytotoxicity, or enhance reactive oxygen species (ROS) generation, nuclear factor (NF)-κB activation, and p-p38 or p21 protein expressions in RAW264.7 macrophage cells. Furthermore, we also separately conjugated two biomolecules containing TAT peptides and bovine serum albumin (BSA) as model systems to study their cell responses in detail. Finally, in vivo mice studies evaluated the biodistribution and blood assays (biochemistry and complete blood count) of PEG-derivative NPs, and results suggested that TAT peptides caused significant white blood cell (WBC)-related cell and platelet abnormalities, as well as liver and kidney dysfunction compared to BSA when conjugated onto MSNs. The results showed that attention to cross-linkers should be considered an issue in the surface modification of NPs. We anticipate that our results could be helpful in developing biosafety nanomaterials.