Photocatalysis, which uses sunlight, N2 and H2O to produce NH3, is a more sustainable approach to N2 fixation than the Haber-Bosch process. However, its efficiency is severely limited by the difficulty of activating NN bonds. This work presents metal (M = Cu, Fe, V)-substituted MIL-125(Ti) (MIL-(MTi)) for photocatalytic N2 fixation without using any sacrificial agents. Structural characterizations reveal that the active sites including oxygen vacancies (OV) and Ti3+ species are formed by the resulting crystal distortion due to the partial substitution of Ti4+ by other metal ions (Cu+, Fe2+, V3+) in MIL-125(Ti). MIL-(CuTi) possesses a larger number of OV and Ti3+ compared to MIL-(FeTi) and MIL-(VTi) due to the larger valence difference between Cu+ and Ti4+. These active sites not only promote the adsorption and activation of N2 and H2O, but also facilitate the photogenerated charge mobility. Photogenerated holes oxidize H2O to produce O2 and H+. Photogenerated electrons reduce N2 activated on Ti3+ sites by combining with H+ to form NH4+. Therefore, MIL-(CuTi) shows the highest NH4+ production rate 46.5 µmol·h−1·g−1, which is much higher than that (1.2 µmol·h−1·g−1) of the pristine MIL-125(Ti). This work provides a new insight into rational design for artificial N2 fixation systems by the construction of the active site.