Highly ordered MCM-41-type mesoporous silica nanoparticles (MSNs) with particle sizes of 150 ± 20 nm were prepared and PEGylated by covalently grafting PEGxk chains of different molecular weights (x = 4, 6, 10, 20) and chain densities (0.05 wt%-3.75 wt%) on the outer surface. The influence of molecular weights and chain densities of PEGxk on the nonspecific binding of PEGylated MSNs to human serum protein (HSA) was investigated. The results revealed that the optimal molecular weights should be not less than 10 k, and the corresponding optimal chain densities for PEG10k-MSNs and PEG20k-MSNs were 0.75 wt% and 0.075 wt%, respectively, and the resultant minimum HSA adsorbance (2.5%) on PEGxk-MSNs was far less than that on MSNs (18.7%) without PEGylation. Under the optimal conditions for the minimum HSA adsorbance, the phagocytosis of human THP-1 monocytic leukemia cell line-derived macrophages (THP-1 macrophages) and the hemolysis of human red blood cells (HRBCs) were investigated with MSNs and PEGylated MSNs. A minimum THP-1 phagocytosis percentage (0.1%) and a very low HRBCs hemolysis percentage (0.9%) of PEG10k-MSNs were obtained, which were much lower than those (8.6% and 14.2%, respectively) of MSNs.