In this study, we demonstrate how functional groups on the surface of mesoporous silica nanoparticles (MSNPs) can influence the encapsulation and release of the anticancer drug doxorubicin, as well as cancer cell response in the absence or presence of serum proteins. To this end, we synthesized four differently functionalized MSNPs with amine, sulfonate, polyethylene glycol, or polyethylene imine functional surface groups, as well as one type of antibody-conjugated MSNP for specific cellular targeting, and we characterized these MSNPs regarding their physicochemical properties, colloidal stability in physiological media, and uptake and release of doxorubicin in vitro. Then, the MSNPs were investigated for their cytotoxic potential on cancer cells. Cationic MSNPs could not be loaded with doxorubicin and did therefore not show any cytotoxic and antiproliferative potential on osteosarcoma cells, although they were efficiently taken up into the cells in the presence or absence of serum. In contrast, substantial amounts of doxorubicin were loaded into negatively charged and unfunctionalized MSNPs. Especially, sulfonate-functionalized doxorubicin-loaded MSNPs were efficiently taken up into the cells in the presence of serum and showed an accelerated toxic and antiproliferative potential compared to unfunctionalized MSNPs, antibody-conjugated MSNPs, and even free doxorubicin. These findings stress the high importance of the surface charge as well as of the protein corona for designing and applying nanoparticles for targeted drug delivery.