The Young’s modulus of single crystalline rutile TiO2 nanoribbons was investigated using nanoindentation. During the experiments, the nanoribbons were laid on three different substrates, including 1 μm thick SiO2 layer on silicon (SiO2/Si), Si(100), and sapphire(0001). Experimental results show the substrates have significant effects on load-indenter displacement curves. To further understand the experimental findings, three-dimensional finite element modeling was carried out to simulate the indentation of nanoribbon-on-substrate systems using ABAQUS. The results show that the receding contact mechanics is a good approximation when describing the contact between the nanoribbon and the substrate. The results also demonstrate that the substrate effect must be carefully considered when performing nanoindentation on one-dimensional nanostructures. Otherwise, the Young’s modulus of the nanostructures could either be overestimated or underestimated. The Young’s modulus is about 360 GPa, comparable to that of bulk TiO2.