Nanoparticles that do not undergo renal excretion or in vivo degradation into biocompatible debris often accumulate in the reticuloendothelial system, also know as the mononuclear phagocyte system, with undesired consequences that limit their clinical utility. In this work, we report the first application of intravital multiphoton fluorescence microscopy to dynamically track the hepatic metabolism of nanoparticles with subcellular resolution in real time. Using fluorescently labeled mesoporous silica nanoparticles (MSNs) in mice as a prototypical model, we observed significant hepatocyte uptake of positively charged, but not negatively charged, moieties. Conversely, in vivo imaging of negatively charged, but not positively charged, MSNs reveals an overwhelming propensity for the former’s rapid uptake by Kupffer cells in liver sinusoids. Since the only prerequisite for these studies was that nanoparticles are fluorescently labeled and not of a specific composition or structure, the techniques we present can readily be extended to a wide variety of nanoparticle structures and surface modifications (e.g., shape, charge, hydrophobicity, PEGylation) in the preclinical assessment and tailoring of their hepatotoxicities and clearances.