Thiamine-coated nanoparticles were prepared by two different preparative methods and evaluated to compare their mucus-penetrating properties and fate in vivo. The first method of preparation consisted of surface modification of freshly poly(anhydride) nanoparticles (NP) by simple incubation with thiamine (T-NPA). The second procedure focused on the preparation and characterization of a new polymeric conjugate between the poly (anhydride) backbone and thiamine prior the nanoparticle formation (T-NPB). The resulting nanoparticles displayed comparable sizes (about 200 nm) and slightly negative surface charges. For T-NPA, the amount of thiamine associated to the surface of the nanoparticles was 15 mu g/mg. For in vivo studies, nanoparticles were labelled with either Tc-99m or Lumogen (R) Red. T-NPA and T-NPB moved faster from the stomach to the small intestine than naked nanoparticles. Two hours post-administration, for T-NPA and T-NPB, > 30% of the given dose was found in close contact with the intestinal mucosa, compared with a 13.5% for NP. Interestingly, both types of thiamine-coated nanoparticles showed a greater ability to cross the mucus layer and interact with the surface of the intestinal epithelium than NP, which remained adhered in the mucus layer. Four hours post-administration, around 35% of T-NPA and T-NPB were localized in the ileum of animals. Overall, both preparative processes yielded thiamine decorated carriers with similar physico-chemical and biodistribution properties, increasing the versatility of these nanocarriers as oral delivery systems for a number of biologically active compounds.