Mesoporous silica nanoparticles (MSNs) have received intensive recent interest as multi-functional nanocarriers for biological cells and tissues. Their cell uptake and intracellular distribution depends on the surface charge they carry. We develop a new approach for monitoring their uptake and intracellular localization by measuring the local pH value of the MSNs. We synthesized a ratiometric pH-sensor loaded with mesoporous silica nanoparticles that can map the location of the MSN through the site pH in HeLa cells. Positively charged MSN-TA was found to locate in higher pH regions which are identified as cytosol mostly. On the other hand, negatively charged MSN-PPs were trapped in acidic endosomes. Evidence is given for direct penetration of membranes through a charge-mediated membrane–nanoparticle interaction mechanism.