E171 (titanium dioxide) is a food additive that has been authorized for use as a food colorant in the European Union. The application of E171 in food has become an issue of debate, since there are indications that it may alter the intestinal barrier. This work applied standardized and validated methodologies to characterize representative samples of 15 pristine E171 materials based on transmission electron microscopy (TEM) and single-particle inductively coupled plasma mass spectrometry (spICP-MS). The evaluation of selected sample preparation protocols allowed identifying and optimizing the critical factors that determine the measurement of the particle size distribution by TEM. By combining optimized sample preparation with method validation, a significant variation in the particle size and shape distributions, the crystallographic structure (rutile versus anatase), and the physicochemical form (pearlescent pigments versus anatase and rutile E171) was demonstrated among the representative samples. These results are important for risk assessment of the E171 food additive and can contribute to the implementation of the European Food Safety Authority (EFSA) guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain.