PEGylated gadolinium oxide nanoparticles (PEG-GdO NPs) as MRI nano-contrast agents (nano-CAs) displayed high relaxivity in our previous study. However, their behaviors in vivo have not been studied systematically yet. Herein, with clinically used CA, Magnevist as control, their toxicity, pharmacokinetics, biodistribution, half-life and excretion in vivo were studied. Mouse experiments after PEG-GdO NP administration, including the analysis of general appearance, histological changes, hepatic and renal functions, were performed to evaluate their toxicity in vivo. MRI and inductively coupled plasma-mass spectrometry (ICP-MS) quantification of Gd accumulation in different organs were introduced to investigate their biodistribution and excretion. The results showed that compared with Magnevist, PEG-GdO NPs presented longer half-life, similar acute toxicity and histological influence, less effect on hepatic and renal functions, and stronger contrast enhancement in tumor, showing their potentials as MRI CA for preclinical applications. Different from kidney clearance of Magnevist, PEG-GdO NPs were mainly excreted via liver.